Methods of proliferating undifferentiated neural cells

ABSTRACT

The invention discloses methods of proliferation and differentiation of multipotent neural stem cells. Also provided are methods of making cDNA libraries and methods of screening biological agents which affect proliferation differentiation survival phenotype or function of CNS cells.

FIELD OF THE INVENTION

[0001] This invention relates to a method for the in vitro culture andproliferation of multipotent neural stem cells, and to the use of thesecells and their progeny as tissue grafts. In one aspect, this inventionrelates to a method for the isolation and in vitro perpetuation of largenumbers of non-tumorigenic neural stem cell progeny which can be inducedto differentiate and which can be used for neurotransplantation in theundifferentiated or differentiated state, into an animal to alleviatethe symptoms of neurologic disease, neurodegeneration and centralnervous system (CNS) trauma. In another aspect, this invention relatesto a method of generating neural cells for the purposes of drugscreening of putative therapeutic agents targeted at the nervous system.In another aspect, this invention also relates to a method of generatingcells for autologous transplantation. In another aspect, the inventionrelates to a method for the in vivo proliferation and differentiation ofthe neural stem cell progeny in the host.

BACKGROUND OF THE INVENTION

[0002] The development of the mammalian central nervous system (CNS)begins in the early stage of fetal development and continues until thepost-natal period. The mature mammalian CNS is composed of neuronalcells (neurons), and glial cells (astrocytes and oligodendrocytes).

[0003] The first step in neural development is cell birth, which is theprecise temporal and spatial sequence in which stem cells and stem cellprogeny (i.e daughter stem cells and progenitor cells) proliferate.Proliferating cells will give rise to neuroblasts, glioblasts and newstem cells.

[0004] The second step is a period of cell type differentiation andmigration when undifferentiated progenitor cells differentiate intoneuroblasts and gliolblasts which give rise to neurons and glial cellswhich migrate to their final positions. Cells which are derived from theneural tube give rise to neurons and glia of the CNS, while cellsderived from the neural crest give rise to the cells of the peripheralnervous system (PNS). Certain factors present during development, suchas nerve growth factor (NGF), promote the growth of neural cells. NGF issecreted by cells of the neural crest and stimulates the sprouting andgrowth of the neuronal axons.

[0005] The third step in development occurs when cells acquire specificphenotypic qualities, such as the expression of particularneurotransmitters. At this time, neurons also extend processes whichsynapse on their targets. Neurons are generated primarily during thefetal period, while oligodendrocytes and astrocytes are generated duringthe early post-natal period. By the late post-natal period, the CNS hasits full complement of nerve cells.

[0006] The final step of CNS development is selective cell death,wherein the degeneration and death of specific cells, fibers andsynaptic connections “fine-tune” the complex circuitry of the nervoussystem. This “fine-tuning” continues throughout the life of the host.Later in life, selective degeneration due to aging, infection and otherunknown etiologies can lead to neurodegenerative diseases.

[0007] Unlike many other cells found in different tissues, thedifferentiated cells of the adult mammalian CNS have little or noability to enter the mitotic cycle and generate new nerve cells. Whileit is believed that there is a limited and slow turnover of astrocytes(Korr et al., J. Comp. Neurol., 150:169, 1971) and that progenitors foroligodendrocytes (Wolsqijk and Noble, Development, 105:386, 1989) arepresent, the generation of new neurons does not normally occur.

[0008] The second step is a period of cell type differentiation andmigration when undifferentiated progenitor cells differentiate intoneuroblasts and gliolbiasts which give rise to neurons and glial cellswhich migrate to their final positions. Cells which are derived from theneural tube give rise to neurons and glia of the CNS, while cellsderived from the neural crest give rise to the cells of the peripheralnervous system (PNS). Certain factors present during development, suchas nerve growth factor (NGF), promote the growth of neural cells. NGF issecreted by cells of the neural crest and stimulates the sprouting andgrowth of the neuronal axons.

[0009] The third step in development occurs when cells acquire specificphenotypic qualities, such as the expression of particularneurotransmitters. At this time, neurons also extend processes whichsynapse on their targets. Neurons are generated primarily during thefetal period, while oligodendrocytes and astrocytes are generated duringthe early post-natal period. By the late post-natal period, the CNS hasits full complement of nerve cells.

[0010] The final step of CNS development is selective cell death,wherein the degeneration and death of specific cells, fibers andsynaptic connections “finetune” the complex circuitry of the nervoussystem. This “finetuning” continues throughout the life of the host.Later in life, selective degeneration due to aging, infection and otherunknown etiologies can lead to neurodegenerative diseases.

[0011] Unlike many other cells found in different tissues, thedifferentiated cells of the adult mammalian CNS have little or noability to enter the mitotic cycle and generate new nerve cells. Whileit is believed that there is a limited and slow turnover of astrocytes(Korr et al., J. Comp. Neurol., 150:169, 1971) and that progenitors foroligodendrocytes (Wolsqijk and Noble, Development, 105:386, 1989) arepresent, the generation of new neurons does not normally occur.Neurogenesis, the generation of new neurons, is complete early in thepostnatal period. However, the synaptic connections involved in neuralcircuits are continuously altered throughout the life of the individual,due to synaptic plasticity and cell death. A few mammalian species (e.g.rats) exhibit the limited ability to generate new neurons in restrictedadult brain regions such as the dentate gyrus and olfactory bulb(Kaplan, J. Comp. Neurol., 195:323, 1981; Bayer, N.Y. Acad. Sci.,457:163, 1985). However, this does not apply to all mammals; and thegeneration of new CNS cells in adult primates does not occur (Rakic,Science, 227:1054, 1985). This inability to produce new nerve cells inmost mammals (and especially primates) may be advantageous for long-termmemory retention; however, it is a distinct disadvantage when the needto replace lost neuronal cells arises due to injury or disease.

[0012] The low turnover of cells in the mammalian CNS together with theinability of the adult mammalian CNS to generate new neuronal cells inresponse to the loss of cells following injury or disease has lead tothe assumption that the adult mammalian CNS does not contain multipotentneural stem cells.

[0013] The critical identifying feature of a stem cell is its ability toexhibit self-renewal or to generate more of itself. The simplestdefinition of a stem cell would be a cell with the capacity forself-maintenance. A more stringent (but still simplistic) definition ofa stem cell is provided by Potten and Loeffler (Development, 110:1001,1990) who have defined stem cells as “undifferentiated cells capable ofa) proliferation, b) self-maintenance, c) the production of a largenumber of differentiated functional progeny, d) regenerating the tissueafter injury, and e) a flexibility in the use of these options.”

[0014] The role of stem cells is to replace cells that are lost bynatural cell death, injury or disease. The presence of stem cells in aparticular type of tissue usually correlates with tissues that have ahigh turnover of cells. However, this correlation may not always hold asstem cells are thought to be present in tissues functions includingmemory.

[0015] Many motor deficits are a result of degeneration in the basalganglia. Huntington's Chorea is associated with the degeneration ofneurons in the striatum, which leads to involuntary jerking movements inthe host. Degeneration of a small region called the subthalamic nucleusis associated with violent flinging movements of the extremities in acondition called ballismus, while degeneration in the putamen and globuspallidus is associated with a condition of slow writhing movements orathetosis. In the case of Parkinson's Disease, degeneration is seen inanother area of the basal ganglia, the substantia nigra par compacta.This area normally sends dopaminergic connections to the dorsal striatumwhich are important in regulating movement. Therapy for Parkinson'sDisease has centered upon restoring dopaminergic activity to thiscircuit.

[0016] Other forms of neurological impairment can occur as a result ofneural degeneration, such as amyotrophic lateral sclerosis and cerebralpalsy, or as a result of CNS trauma, such as stroke and epilepsy.

[0017] Demyelination of central and peripheral neurons occurs in anumber of pathologies and leads to improper signal conduction within thenervous systems. Myelin is a cellular sheath, formed by glial cells,that surrounds axons and axonal processes that enhances variouselectrochemical properties and provides trophic support to the neuron.Myelin is formed by Schwann cells in the PNS and by oligodendrocytes inthe CNS. Among the various demyelinating diseases MS is the mostnotable.

[0018] To date, treatment for CNS disorder has been primarily via theadministration of pharmaceutical compounds. Unfortunately, this type oftreatment has been fraught with many complications including the limitedability to transport drugs across the blood-brain barrier and thedrug-tolerance which is acquired by patients to whom these drugs areadministered long-term. For instance, partial restoration ofdopaminergic activity in Parkinson's patients has been achieved withlevodopa, which is a dopamine precursor able to cross the blood-brainbarrier. However, patients become tolerant to the effects of levodopa,and therefore, steadily increasing dosages are needed to maintain itseffects. In addition, there are a number of side effects associated withlevodopa such as increased and uncontrollable movement.

[0019] Recently, the concept of neurological tissue grafting has beenapplied to the treatment of neurological diseases such as Parkinson'sDisease. Neural grafts may avert the need not only for constant drugadministration, but also for complicated drug delivery systems whicharise due to the blood-brain barrier. However, there are limitations tothis technique as well. First, cells used for transplantation whichcarry cell surface molecules of a differentiated cell from another hostcan induce an immune reaction in the host. In addition, the cells mustbe at a stage of development where they are able to form normal neuralconnections with neighboring cells. For these reasons, initial studieson neurotransplantation centered on the use of fetal cells. Perlow, etal. describe the transplantation of fetal dopaminergic neurons intoadult rats with chemically induced nigrostriatal lesions in “Braingrafts reduce motor abnormalities produced by destruction ofnigrostriatal dopamine system,” Science 204:643-647 (1979). These graftsshowed good survival, axonal outgrowth and significantly reduced themotor abnormalities in the host animals.

[0020] In both human demyelinating diseases and rodent models there issubstantial evidence that demyelinated neurons are capable ofremyelination in vivo. In MS, for example, it appears that there areoften cycles of de- and remyelination. Similar observations in rodentdemyelinating paradigms lead to the prediction that exogenously appliedcells would be capable of remyelinating demyelinated axons. Thisapproach has proven successful in a number of experimental conditions[Freidman et al., Brain Research, 378:142-146 (1986); Raine, et al.,Laboratory Investigation 59:467476 (1988); Duncan et al., J. ofNeurocytology, 17:351-360 (1988)]. The sources of cells for some ofthese experiments included dissociated glial cell suspensions preparedfrom spinal cords (Duncan et al., supra), Schwann cell cultures preparedfrom sciatic nerve [Bunge et al., 1992, WO 92/03536; Blakemore andCrang, J. Neurol. Sci., 70:207-223 (1985)]; cultures from dissociatedbrain tissue [Blakemore and Crang, Dev. Neurosci. 10:1-11 (1988)],oligodendrocyte precursor cells [Gumpel et al., Dev. Neurosci.11:132-139 (1989)], O-2A cells [Wolswijk et al., Development 109:691-608(1990); Raff et al., Nature 3030:390-396 (1983); Hardy et al.,Development 111:1061-1080 (1991)], and immortalized O-2A cell lines,[Almazan and McKay Brain Res. 579:234-245 (1992)].

[0021] O-2A cells are glial progenitor cells which give rise in vitroonly to oligodendrocytes and type II astrocytes. Cells which appear byimmunostaining in vivo to have the O-2A phenotype have been shown tosuccessfully remyelinate demyelinated neurons in vivo, [Godfraind etal., J. Cell Biol. 109:2405-2416 (1989)]. Injection of a large number ofO-2A cells is required to adequately remyelinate all targeted neurons invivo, since it appears that O-2A cells (like other glial cellpreparations) do not continue to divide in vivo. Although O-2Aprogenitor cells can be grown in culture, currently the only availableisolation technique employs optic nerve as starting material. This is alow yield source, which requires a number of purification steps. Thereis an additional drawback that O-2A cells isolated by the availableprocedures are capable of only a limited number of divisions [RaffScience 243:1450-1455 (1989)].

[0022] Although adult CNS neurons are not good candidates forneurotransplantation, neurons from the adult PNS have been shown tosurvive transplantation, and to exert neurotrophic and gliotrophiceffects on developing host neural tissue. One source of non-CNS neuraltissue for transplantation is the adrenal medulla. Adrenal chromaffincells originate from the neural crest like PNS neurons, and receivesynapses and produce carrier and enzyme proteins similar to PNS neurons.Although these cells function in an endocrine manner in the intactadrenal medulla, in culture these cells lose their glandular phenotypeand develop certain neural features in culture in the presence ofcertain growth factors and hormones [Notter, et al., “Neuronalproperties of monkey adrenal medulla in vitro, Cell Tissue Research244:69-76 (1986)]. When grafted into mammalian CNS, these cells surviveand synthesize significant quantities of dopamine which can interactwith dopamine receptors in neighboring areas of the CNS.

[0023] In U.S. Pat. No. 4,980,174, transplantation ofmonoamine-containing cells isolated from adult rat pineal gland andadrenal medulla into rat frontal cortex led to the alleviation oflearned helplessness, a form of depression in the host. In U.S. Pat. No.4,753,635, chromaffin cells and adrenal medullary tissue derived fromsteers were implanted into the brain stem or spinal cord of rats andproduced analgesia when the implanted tissue or cell was induced torelease nociceptor interacting substances (i.e. catecholamines such asdopamine). Adrenal medullary cells have been autologously grafted intohumans, and have survived, leading to mild to moderate improvement insymptoms (Watts, et al., “Adrenal-caudate transplantation in patientswith Parlinson's Disease (PD): 1-year follow-up,” Neurology 39 Suppl 1:127 [1989], Hurtig, et al., “Postmortem analysis ofadrenal-medulla-to-caudate autograft in a patient with Parkinson'sDisease,” Annals of Neurology 25: 607-614 [1989]). However, adrenalcells do not obtain a normal neural phenotype, and are thereforeprobably of limited use for transplants where synaptic connections mustbe formed.

[0024] Another source of tissue for neurotransplantation is from celllines. Cell lines are immortalized cells which are derived either bytransformation of normal cells with an oncogene (Cepko, “Immortalizationof neural cells via retrovirus-mediated oncogene transduction,” Ann.Rev. Neurosci. 12:47-65 [1989]) or by the culturing of cells withaltered growth characteristics in vitro (Ronnett, et al., “Humancortical neuronal cell line: Establishment from a patient withunilateral megalencephaly,” Science 248:603-605 [1990]). Such cells canbe grown in culture in large quantities to be used for multipletransplantations. Some cell lines have been shown to differentiate uponchemical treatment to express a variety of neuronal properties such asneurite formation, excitable membranes and synthesis ofneurotransmitters and their receptors. Furthermore, upondifferentiation, these cells appear to be amitotic, and thereforenoncancerous. However, the potential for these cells to induce adverseimmune responses, the use of retroviruses to immortalize cells, thepotential for the reversion of these cells to an amitotic state, and thelack of response of these cells to normal growth-inhibiting signals makecell lines less than optimal for widespread use.

[0025] Another approach to neurotransplantation involves the use ofgenetically engineered cell types or gene therapy. Using this method, aforeign gene or transgene can be introduced into a cell which isdeficient in a particular enzymatic activity, thereby allowing the cellto express the gene. Cells which now contain the transferred gene can betransplanted to the site of neurodegeneration, and provide products suchas neurotransmitters and growth factors (Rosenberg, et al., “Graftinggenetically modified cells to the damaged brain: Restorative effects ofNGF Expression,” Science 242:1575-1578, [1988]) which may function toalleviate some of the symptoms of degeneration. However, there stillexists a risk of inducing an immune reaction using currently availablecell lines. In addition, these cells may also not achieve normalneuronal connections with the host tissue.

[0026] Genetically modified cells have been used in neurological tissuegrafting in order to replace lost cells which normally produce aneurotransmitter. For example, fibroblasts have been geneticallymodified with a retroviral vector containing a cDNA for tyrosinehydroxylase, which allows them to produce dopamine, and implanted intoanimal models of Parkinson's Disease (Gage et al., U.S. Pat. No.5,082,670).

[0027] While the use of genetically modified fibroblasts to treat CNSdisorders has shown promise in improving some behavioral deficits inanimal models of Parkinson's Disease, and represents a novel approach tosupplying a needed transmitter to the CNS, it suffers from severalsignificant drawbacks as a treatment for Parkinson's Disease and ingeneral as a therapeutic approach for treating neurodegenerativediseases and brain injury. First, the CNS is primarily composed of threecell types—neurons, astrocytes and oligodendrocytes. The implantation ofa foreign cell such as a fibroblast into the CNS and its direct andindirect effects on the functioning of the host cells has yet to bestudied. However, it is likely that the expression of membrane boundfactors and the release of soluble molecules such as growth factors andproteases will alter the normal behavior of the surrounding tissue. Thismay result in the disruption of neuronal firing patterns either by adirect action on neurons or by an alteration in the normal functioningof glial cells.

[0028] Another concern that arises when fibroblasts are implanted intothe CNS is the possibility that the implanted cells may lead to tumorformation because the intrinsic inhibition of fibroblast division ispoorly controlled. Instead, extrinsic signals play a major role incontrolling the number of divisions the cell will undergo. The effect ofthe CNS environment on the division of implanted fibroblasts and thehigh probability of a fibroblastic tumor formation has not been studiedin the long-term.

[0029] A third concern in transplanting fibroblasts into the CNS is thatfibroblasts are unable to integrate with the CNS cells as astrocytes,oligodendrocytes, or neurons do. Fibroblasts are intrinsically limitedin their ability to extend neuronal-like processes and form synapseswith host tissue. Hence, although the genetic modification andimplantation of fibroblasts into the CNS represents an improvement overthe current technology for the delivery of certain molecules to the CNS,the inability of fibroblasts to integrate and function as CNS tissue,their potential negative effects on CNS cells, and their limitedintrinsic control of proliferation limits their practical usage forimplantation for the treatment of acute or chronic CNS injury ordisease.

[0030] A preferred tissue for genetic modification and implantationwould be CNS cells neurons, astrocytes, or oligodendrocytes. One sourceof CNS cells is from human fetal tissue. Several studies have shownimprovements in patients with Parldnson's Disease after receivingimplants of fetal CNS tissue. Implants of embryonic mesencephalic tissuecontaining dopamine cells into the caudate and putamen of human patientswas shown by Freed et al. (N Engl J Med 327:1549-1555 (1992)) to offerlong-term clinical benefit to some patients with advanced Parkinson'sDisease. Similar success was shown by Spencer et al. (N Engl J Med327:1541-1548 (1992)). Widner et al. (N Engl J Med 327:1556-1563 (1992))have shown long-term functional improvements in patients withMPTP-induced Parkinsonism that received bilateral implantation of fetalmesencephalic tissue.

[0031] While the studies noted above are encouraging, the use of largequantities of aborted fetal tissue for the treatment of disease raisesethical considerations and political obstacles. There are otherconsiderations as well. Fetal CNS tissue is composed of more than onecell type, and thus is not a well-defined source of tissue. In addition,there are serious doubts as to whether an adequate and Constant supplyof fetal tissue would be available for transplantation. For example, inthe treatment of MPTP-induced Parkinsonism (Widner supra) tissue from 6to 8 fetuses were used for implantation into the brain of a singlepatient. There is also the added problem of the potential forcontamination during fetal tissue preparation. Moreover, the tissue mayalready be infected with a bacteria or virus, thus requiring expensivediagnostic testing for each fetus used. However, even diagnostic testingmight not uncover all infected tissue. For example, the diagnosis ofHIV-free tissue is not guaranteed because antibodies to the virus aregenerally not present until several weeks after infection.

[0032] While currently available transplantation approaches represent asignificant improvement over other available treatments for neurologicaldisorders, they suffer from significant drawbacks. The inability in theprior art of the transplant to fully integrate into the host tissue, andthe lack of availability of cells in unlimited amounts from a reliablesource for grafting are, perhaps, the greatest limitations ofneurotransplantation.

[0033] It would be more preferable to have a well-defined, reproduciblesource of neural tissue for transplantation that is available inunlimited amounts. Since adult neural tissue undergoes minimal division,it does not readily meet these criteria. While astrocytes retain theability to divide and are probably amenable to infection with foreigngenes, their ability to form synapses with neuronal cells is limited andconsequently so is their extrinsic regulation of the expression andrelease of the foreign gene product.

[0034] Oligodendrocytes suffer from some of the same problems. Inaddition, mature oligodendrocytes do not divide, limiting the infectionof oligodendrocytes to their progenitor cells (e.g. O2A cells). However,due to the limited proliferative ability of oligodendrocyte progenitors,the infection and harvesting of these cells does not represent apractical source.

[0035] The infection of neurons with foreign genes and implantation intothe CNS would be ideal due to their ability to extend processes, makesynapses and be regulated by the environment. However, differentiatedneurons do not divide and transfection with foreign genes by chemicaland physical means is not efficient, nor are they stable for longperiods of time. The infection of primary neuronal precursors withretroviral vectors in vitro is not practical either because neuroblastsare intrinsically controlled to undergo a limited number of divisionsmaking the selection of a large number of neurons, that incorporate andexpress the foreign gene, nearly impossible. The possibility ofimmortalizing the neuronal precursors by retroviral transfer ofoncogenes and their subsequent infection of a desired gene is notpreferred due to the potential for tumor formation by the implantedcells.

[0036] In addition to the need for a well-defined, reproducible sourceof neural cells available in unlimited amounts for transplantationpurposes, a similar need exists for drug screening purposes and for thestudy of CNS function, dysfunction, and development. The mature humannervous system is composed of billions of cells that are generatedduring development from a small number of precursors located in theneural tube. Due to the complexity of the mammalian CNS, the study ofCNS developmental pathways, as well as alterations that occur in adultmammalian CNS due to dysfunction, has been difficult. Such areas wouldbe better studied using relatively simple models of the CNS underdefined conditions.

[0037] Generally, two approaches have been taken for studying culturedCNS cells: the use of primary neural cultures; and the use of neuralcell lines. Primary mammalian neural cultures can be generated fromnearly all brain regions providing that the starting material isobtained from fetal or early post-natal animals. In general, three typesof cultures can be produced, enriched either in neurons, astrocytes, oroligodendrocytes. Primary CNS cultures have proven valuable fordiscovering many mechanisms of neural function and are used for studyingthe effects of exogenous agents on developing and mature cells. Whileprimary CNS cultures have many advantages, they suffer from two primarydrawbacks. First, due to the limited proliferative ability of primaryneural cells, new cultures must be generated from several differentanimals. While great care is usually taken to obtain tissue at identicalstates of development and from identical brain regions, it is virtuallyimpossible to generate primary cultures that are identical. Hence, thereexists a significant degree of variability from culture to culture.

[0038] A second disadvantage of primary cultures is that the tissue mustbe obtained from fetuses or early post-natal animals. If primarycultures are to be performed on a regular basis, this requires theavailability of a large source of starting material. While this isgenerally not a problem for generating primary cultures from somespecies (e.g. rodents), it is for others (e.g. primates). Due to thelimited supply and ethical concerns, the culturing of primary cells fromprimates (both human and non-human) is not practical.

[0039] Due to the limited proliferative ability of primary neural cells,the generation of a large number of homogenous cells for studies ofneural function, dysfunction, and drug design/screening has previouslynot been achieved. Therefore, homogenous populations of cells that cangenerate a large number of progeny for the in vitro investigation of CNSfunction has been studied by the use of cell lines. The generation ofneural cell lines can be divided into two categories: 1) spontaneouslyoccurring tumors, and 2) custom-designed cell lines.

[0040] Of the spontaneously occurring tumors, probably the most studiedcell line for neurobiology is the rat pheochromocytoma (PC12) cells thatcan differentiate into sympathetic-like neurons in response to NGF.These cells have proven to be a useful model for studying mechanisms ofneural development and alterations (molecular and cellular) in responseto growth factors. Neuroblastoma and glioma cell lines have been used tostudy neuronal and glial functioning [Liles, et al., J. Neurosci. 7,2556-2563 (1987); Nister et al. Cancer Res. 48(14) 3910 (1988)].Embryonal carcinoma cells are derived from teratoma tumors of fetal germcells and have the ability to differentiate into a large number ofnon-neural cell types with some lines (e.g. P19 cells) [Jones-Villeneuveet al. J. Cell Biol. 94, 253-262 (1982)] having the ability todifferentiate into neural cells [(McBurney et al. J. Neurosci. 8(3)1063-73 (1993)]. A human teratocarcinoma-derived cell line, NTera2/c1.D1, with a phenotype resembling CNS neuronal precursor cells, canbe induced to differentiate in the presence of retinoic acid. However,the differentiated cells are restricted to a neuronal phenotype[Pleasure and Lee J. Neurosci. Res. 35: 585-602 (1993)]. While thesetypes of cell lines are able to generate a large number of cells forscreening the effects of exogenous agents on cell survival or function,the limited number of these types of lines, the limited number ofphenotypes that they are able to generate and the unknown nature oftheir immortalization (which may effect the function of the cells in anundefined manner) makes these types of cell lines less than ideal for invitro models of neural function and discovery of novel therapeutics.

[0041] An alternative approach to spontaneously occurring cell lines isthe intentional immortalization of a primary cell by introducing anoncogene that alters the genetic make-up of the cell thereby inducingthe cell to proliferate indefinitely. This approach has been used bymany groups to generate a number of interesting neural cell lines[(Bartlett et al. Proc. Nat. Acad. Sci. 85(9) 3255-3259 (1988);Frederiksen et al. Neuron 1, 439-448 (1988); Trotter et al. Oncogene 4:457464 (1989); Ryder et al. J. Neurobiol. 21: 356-375 (1980); Murphy etal. J. Neurobiol 22: 522-535 (1991); Almazan and McKay et al. Brain Res.579: 234-245 (1992)]. While these lines may prove useful for studyingthe decisions that occur during cell determination and differentiation,and for testing the effects of exogenous agents, they suffer fromseveral drawbacks. First, the addition of an oncogene that alters theproliferative status of a cell may affect other properties of the cell(oncogenes may play other roles in cells besides regulating the cellcycle). This is well illustrated in a study by Almazan and McKay, supra,and their immortalization of an oligodendrocyte precursor from the opticnerve which is unable to differentiate into type II astrocytes(something that normal optic nerve oligodendrocyte precursors can do).The authors suggest the presence of the immortalizing antigen may alterthe cells ability to differentiate into astrocytes.

[0042] Another drawback to using intentionally immortalized cellsresults from the fact that the nervous system is composed of billions ofcells and possibly thousands of different cell types, each with uniquepatterns of gene expression and responsiveness to their environment. Acustom-designed cell line is the result of the immortalization of asingle progenitor cell and its clonal expansion. While a large supply ofone neural cell type can be generated, this approach does not take intoaccount cellular interactions between different cell types. In addition,while it is possible to immortalize cells from a given brain region,immortalization of a desired cell is not possible due to the lack ofcontrol over which cells will be altered by the oncogene. Hence, whilecustom designed cell lines offer a few advantages over spontaneouslyoccurring tumors, they suffer from several drawbacks and are less thanideal for understanding CNS function and dysfunction.

[0043] Therefore, in view of the aforementioned deficiencies attendantwith prior art methods of neural cell culturing, transplantation, andCNS models, a need exists in the art for a reliable source of unlimitednumbers of undifferentiated neural cells for neurotransplantation anddrug screening which are capable of differentiating into neurons,astrocytes, and oligodendrocytes. Preferably cellular division in suchcells from such a source would be epigenetically regulated and asuitable number of cells could be efficiently prepared in sufficientnumbers for transplantation. The cells should be suitable in autografts,xenografts, and allografts without a concern for tumor formation. Thereexists a need for the isolation, perpetuation and transplantation ofautologous neural cells from the juvenile or adult brain that arecapable of differentiating into neurons and glia.

[0044] A need also exists for neural cells, capable of differentiatinginto neurons, astrocytes and oligodendrocytes that are capable ofproliferation in vitro and thus amenable to genetic modificationtechniques.

[0045] Additionally, there exists a need for the repair of damagedneural tissue in a relatively non-invasive fashion, that is by inducingneural cells to proliferate and differentiate into neurons, astrocytes,and oligodendrocytes in vivo, thereby averting the need fortransplantation.

[0046] Accordingly, a major object of the present invention is toprovide a reliable source of an unlimited number of neural cells forneurotransplantation that are capable of differentiating into neurons,astrocytes, and oligodendrocytes.

[0047] It is another object of the present invention to provide a methodfor the in vitro proliferation of neural stem cells from embryonic,juvenile and adult brain tissue, to produce unlimited numbers ofprecursor cells available for transplantation that are capable ofdifferentiating into neurons, astrocytes, and oligodendrocytes.

[0048] A further object of the invention is to provide methods forinducing neural cells to proliferate and differentiate in vivo, therebyaverting the need for neurotransplantation.

[0049] A still further object of the invention is to provide a method ofgenerating large numbers of normal neural cells for the purpose ofscreening putative therapeutic agents targeted at the nervous system andfor models of CNS development, function, and dysfunction.

SUMMARY OF THE INVENTION

[0050] This invention provides in one aspect a composition for inducingthe proliferation of a multipotent neural stem cell comprising a culturemedium supplemented with at least one growth factor, preferablyepidermal growth factor or transforming growth factor alpha.

[0051] The invention also provides a method for the in vitroproliferation and differentiation of neural stem cells and stem cellprogeny comprising the steps of (a) isolating the cell from a mammal,(b) exposing the cell to a culture medium containing a growth factor,(c) inducing the cell to proliferate, and (d) inducing the cell todifferentiate. Proliferation and perpetuation of the neural stem cellprogeny can be carried out either in suspension cultures, or by allowingcells to adhere to a fixed substrate. Proliferation and differentiationcan be done before or after transplantation, and in various combinationsof in vitro or in vivo conditions, including (1) proliferation anddifferentiation in vitro, then transplantation, (2) proliferation invitro transplantation, then further proliferation and differentiation invivo, and (3) proliferation in vitro, transplantation anddifferentiation in vivo. The invention also provides for theproliferation and differentiation of the progenitor cells in vivo, whichcan be done directly in the host without the need for transplantation.

[0052] The invention also provides a method for the in vivotransplantation of neural stem cell progeny, treated as in any of (1)through (3) above, which comprises implanting, into a mammal, thesecells which have been treated with at least one growth factor.

[0053] Furthermore, the invention provides a method for treatingneurodegenerative diseases comprising administering to a mammal neuralstem cell progeny which have been treated as in any of (1) through (3),and induced to differentiate into neurons and/or glia.

[0054] The invention also provides a method for treatingneurodegenerative disease comprising stimulating in vivo mammalian CNSneural stem cells to proliferate and the neural stem cell progeny todifferentiate into neurons and/or glia.

[0055] The invention also provides a method for the transfection ofneural stem cells and stem cell progeny with vectors which can expressthe gene products for growth factors, growth factor receptors, andpeptide neurotransmitters, or express enzymes which are involved in thesynthesis of neurotransmitters, including those for amino acids,biogenic amines and neuropeptides, and for the transplantation of thesetransfected cells into regions of neurodegeneration.

[0056] In a still further aspect, the invention provides a method forthe screening of potential neurologically therapeutic pharmaceuticalsusing neural stem cell progeny which have been proliferated in vitro.

BRIEF DESCRIPTION OF THE FIGURES

[0057]FIG. 1: Diagram Illustrating the Proliferation of a MultipotentNeural Stem Cell

[0058] (A) In the presence of a proliferation-inducing growth factor thestem cell divides and gives rise to a sphere of undifferentiated cellscomposed of more stem cells and progenitor cells. (B) When the clonallyderived sphere of undifferentiated cells is dissociated and plated assingle cells, on a non-adhesive substrate and in the presence of aproliferation-inducing growth factor, each stem cell will generate a newsphere. (C) If the spheres are cultured in conditions that allowdifferentiation, the progenitor cells differentiate into neurons,astrocytes and oligodendrocytes.

[0059]FIG. 2: Proliferation of Epidermal Growth Factor (EGF) ResponsiveCells

[0060] After 2 days in vitro EGF-responsive cells begin to proliferate(FIG. 2A). After 4 days in vitro small clusters of cells known asneurospheres are apparent (FIG. 2B). The neurospheres of continuouslyproliferating cells continue to grow in size (FIG. 2C) until they liftoff the substrate and float in suspension (FIG. 2D). At this stage, thefloating spheres can be easily removed, dissociated into single cellsand, in the presence of EGF, proliferation can be re-initiated. (Bar: 50μm).

[0061]FIG. 3: Differentiation of Cells from Single EGF-Generated Spheresinto Neurons, Astrocytes, and Oligodendrocytes

[0062] Triple-label immunocytochemistry with antibodies to microtubuleassociated protein (MAP-2), glial fibrillary acidic protein (GFAP), andO4 (a cell surface antigen) are used to detect the presence of neurons(FIG. 3B), astrocytes (FIG. 3C) and oligodendrocytes (FIG. 3D),respectively, from an EGF-generated, stem cell-derived neurosphere (FIG.3A) derived from primary culture. (Bar: 50 μm).

DETAILED DESCRIPTION OF THE INVENTION

[0063] The present invention provides methods for inducing multipotentneural stem cells from fetal, juvenile, or adult mammalian tissue toproliferate in vitro or in vivo (i.e. in situ), to generate largenumbers of neural stem cell progeny capable of differentiating intoneurons, astrocytes, and oligodendrocytes. Methods for differentiationof the neural stem cell progeny are also provided. The induction ofproliferation and differentiation of neural stem cells can be doneeither by culturing the cells in suspension or on a substrate onto whichthey can adhere. Alternatively, proliferation and differentiation ofneural stem cells can be induced, under appropriate conditions, in thehost in the following combinations: (1) proliferation anddifferentiation in vitro, then transplantation, (2) proliferation invitro, transplantation, then further proliferation and differentiationin vivo, (3) proliferation in vitro, transplantation and differentiationin vivo, and (4) proliferation and differentiation in vivo.Proliferation and differentiation in vivo (i.e. in situ) can involve anon-surgical approach that coaxes neural stem cells to proliferate invivo with pharmaceutical manipulation. Thus, the invention provides ameans for generating large numbers of undifferentiated anddifferentiated neural cells for neurotransplantation into a host inorder to treat neurodegenerative disease and neurological trauma, fornon-surgical methods of treating neurodegenerative disease andneurological trauma, and for drug-screening applications.

[0064] Multipotent Neural Stem Cells

[0065] Neurobiologists have used various terms interchangeably todescribe the undifferentiated cells of the CNS. Terms such as “stemcell”, “precursor cell” and “progenitor cell” are commonly used in thescientific literature. However, there are different types ofundifferentiated neural cells, with differing characteristics and fates.U.S. Ser. No. 08/270,412 which is a continuation application of U.S.Ser. No. 07/726,812, termed the cells obtained and proliferated usingthe methods of Examples 1-4 below “progenitor cells”. The terminologyused for undifferentiated neural cells has evolved such that these cellsare now termed “neural stem cells”. U.S. Ser. No. 08/270,412 defines the“progenitor” cell proliferated in vitro to mean “an oligopotent ormultipotent stem cell which is able to divide without limit and underspecific conditions can produce daughter cells which terminallydifferentiate into neurons and glia.” The capability of a cell to dividewithout limit and produce daughter cells which terminally differentiateinto neurons and glia are stem cell characteristics. Accordingly, asused herein, the cells proliferated using the methods described inExamples 1-4 are termed “neural stem cells”. A neural stem cell is anundifferentiated neural cell that can be induced to proliferate usingthe methods of the present invention. The neural stem cell is capable ofself-maintenance, meaning that with each cell division, one daughtercell will also be a stem cell. The non-stem cell progeny of a neuralstem cell are termed progenitor cells. The progenitor cells generatedfrom a single multipotent neural stem cell are capable ofdifferentiating into neurons, astrocytes (type I and type II) andoligodendrocytes. Hence, the neural stem cell is “multipotent” becauseits progeny have multiple differentiative pathways.

[0066] The term “neural progenitor cell”, as used herein, refers to anundifferentiated cell derived from a neural stem cell, and is not itselfa stem cell. Some progenitor cells can produce progeny that are capableof differentiating into more than one cell type. For example, an O-2Acell is a glial progenitor cell that gives rise to oligodendrocytes andtype II astrocytes, and thus could be termed a “bipotential” progenitorcell. A distinguishing feature of a progenitor cell is that, unlike astem cell, it has limited proliferative ability and thus does notexhibit self-maintenance. It is committed to a particular path ofdifferentiation and will, under appropriate conditions, eventuallydifferentiate into glia or neurons.

[0067] The term “precursor cells”, as used herein, refers to the progenyof neural stem cells, and thus includes both progenitor cells anddaughter neural stem cells.

[0068] Neural stem cell progeny can be used for transplantation into aheterologous, autologous, or xenogeneic host. Multipotent neural stemcells can be obtained from embryonic, post-natal, juvenile or adultneural tissue. The neural tissue can be obtained from any animal thathas neural tissue such as insects, fish, reptiles, birds, amphibians,mammals and the like. The preferred source neural tissue is frommammals, preferably rodents and primates, and most preferably, mice andhumans.

[0069] In the case of a heterologous donor animal, the animal may beeuthanized, and the neural tissue and specific area of interest removedusing a sterile procedure. Areas of particular interest include any areafrom which neural stem cells can be obtained that will serve to restorefunction to a degenerated area of the host's nervous system,particularly the host's CNS. Suitable areas include the cerebral cortex,cerebellum, midbrain, brainstem, spinal cord and ventricular tissue, andareas of the PNS including the carotid body and the adrenal medulla.Preferred areas include regions in the basal ganglia, preferably thestriatum which consists of the caudate and putamen, or various cellgroups such as the globus pallidus, the subthalamic nucleus, the nucleusbasalis which is found to be degenerated in Alzheimer's Diseasepatients, or the substantia nigra pars compacta which is found to bedegenerated in Parkinson's Disease patients. Particularly preferredneural tissue is obtained from ventricular tissue that is found liningCNS ventricles and includes the subependyma. The term “ventricle” refersto any cavity or passageway within the CNS through which cerebral spinalfluid flows. Thus, the term not only encompasses the lateral, third, andfourth ventricles, but also encompasses the central canal, cerebralaqueduct, and other CNS cavities.

[0070] Human heterologous neural stem cells may be derived from fetaltissue following elective abortion, or from a post-natal, juvenile oradult organ donor. Autologous neural tissue can be obtained by biopsy,or from patients undergoing neurosurgery in which neural tissue isremoved, for example, during epilepsy surgery, temporal lobectomies andhippocampalectomies. Neural stem cells have been isolated from a varietyof adult CNS ventricular regions, including the frontal lobe, conusmedullaris, thoracic spinal cord, brain stem, and hypothalamus, andproliferated in vitro using the methods detailed herein. In each ofthese cases, the neural stem cell exhibits self-maintenance andgenerates a large number of progeny which include neurons, astrocytesand oligodendrocytes.

[0071] Normally, the adult mammalian CNS is mitotically quiescent invivo with the exception of the subependymal region lining the lateralventricles in the forebrain. This region contains a subpopulation ofconstitutively proliferating cells with a cell cycle time of 12.7 hours.BrdU and retroviral labeling of the proliferating cells reveal that noneof the newly generated cells differentiate into mature neurons or glianor do they migrate into other CNS regions (Morshead and Van der Kooy,supra).

[0072] The continual proliferation and maintenance of a constant numberof cells within the subependyma is explained by two mechanisms. Thedeath of one of the daughter cells after each division maintains theproliferating population at a constant number. The constitutivelydividing population eventually dies out (and hence is not a stem cellpopulation) however, a subpopulation of relatively quiescent cellswithin the subependyma is able to repopulate the Constitutively dividingpopulation. This stem cell-like mode of maintaining the proliferativesubependymal population is analogous to other tissues where cells have ashort life span and are repopulated by a subpopulation of relativelyquiescent cells referred to as stem cells.

[0073] As detailed in Example 27, experiments utilizing retrovirusinfection of constituitively proliferating cells in vivo and subsequentβ-galactosidase (β-gal) reporter gene expression as a non-dilutingmarker show that with increasing adult mice survival times (of up to 28days post retrovirus infection) there is a progressive loss of β-galpositive subependymal cells. Relative to 1 day survival animals, 6 daysfollowing retrovirus injection there is a 45% loss of β-gal positivecells and 28 days following retrovirus infection there is a 97% loss.Using nested polymerase chain reaction (PCR) to identify single cellscontaining retroviral DNA it was determined that the loss of β-galexpressing cells is due to the loss of the retrovirally infected cellsthrough cell death, not due to the turn-off of β-gal expression.

[0074] Intraperitoneal injections of BrdU (a thymidine analog that isincorporated into the DNA of dividing cells) reveal that 33% of thecells within some regions of the subependyma make up the normallyconstituitively dividing population (see Morshead and van der Kooy, J.Neurosci. 12:249 (1992)). The number of BrdU labelled cells decreasesover time. By 30 days after BrdU labeling, only 3% of the dividing cellsare still labelled. The heavy labeling of only a small number of cells30 days after BrdU injections demonstrates that although the labelledcells were dividing at the time of the injections they were relativelyquiescent for the 30 day period. This suggests that these few labeledcells are stem cells rather than cells of the constitutivelyproliferating population.

[0075] The above two examples support the hypothesis that themaintenance of the constant number of proliferating subependymal cellsseen throughout adult life requires the presence of a relativelyquiescent stem cell that proliferates sporadically to replenish theconstitutively proliferating population and to self-renew.

[0076] As detailed in Example 24, the constitutively dividingsubependymal cells can be killed off by injecting high doses ofradioactive thymidine for the duration of the cell cycle at intervalsless than S-phase duration. At one day post-kill the proliferatingpopulation is 10% of controls and by 8 days the proliferating populationis back to control levels. If the replenished population is due to therecruitment of normally quiescent stem cells into the proliferativemode, then a second kill at the time that stem cells are generatingprogeny to repopulate the subependyma should alter the number of cellswithin the constitutively proliferating population. When a second killis done 2 days after the initial kill, 8 days later the constitutivelyproliferating population is only 45% of the control values (animalsreceiving no thymidine kill treatment) or animals that received only onekill at day 0 (the time of the first kill). The reduction in the numberof proliferative cells in the subependyma is maintained at 63% even at31 days after the second kill. When a second kill is done on day 4, theproliferating population returns to 85% of control values 8 days later.These results suggest that the normally quiescent stem cell is recruitedinto the proliferative mode within the first two days after the initialkill and that by 4 days the stem cell no longer needs to be recruited torepopulate the subependyma.

[0077] As detailed in Example 26 below, an experiment was performed todetermine whether the in vitro stem cell is derived from theconstitutively proliferating population or from the quiescentpopulation. Animals were treated in one of the following ways: Group 1.Control High doses of radioactive thymidine were given on: Group 1.Control High doses of radioactive thymidine were given on: Group 2. day0 Group 3. day 0 and day 2 Group 4. day 0 and day 4

[0078] 16 to 20 following the last injection animals were killed andstem cells isolated from the striatum (including the subependymalregion) via the methods described in Example 2 below.

[0079] In groups 2-4 the constitutively proliferating population waskilled. In group 3 stem cells that are recruited into the cell cycle torepopulate the subependymal proliferating cells were also killed. Numberof Neurospheres produced in vitro: Number of Neurospheres produced invitro: Group 1. 100% (Control) Group 2. 100% Group 3.  45% Group 4.  85%

[0080] These results demonstrate that when you eliminate nearly all ofthe constitutively proliferating cells in the subependyma this does notaffect the number of stem cells that can be isolated and proliferated invitro (group 1 vs. group 2 and 4). However, when the normally quiescentcells are killed when they are recruited to repopulate the subependyma(as with group 3) the number of stem cells that can be isolated in vitrois significantly reduced (group 3 vs. group 1 and 2). By 4 days afterthe first kill most of the stem cells themselves are no longer turningover and as a result are not killed by the second series of tritiatedthymidine injections (hence, only a 15% reduction [group 4] compared to55% reduction [group 3]).

[0081] The above results demonstrate that, in adult, the stem cellswhich are proliferated in vitro are derived from the quiescentpopulation of subependymal cells in vivo. This also explains why stemcells can be derived from CNS ventricular regions, other than theforebrain, which do not have a subpopulation of constitutivelyproliferating cells.

[0082] In vitro Proliferation of Neutral Stem Cells

[0083] Cells can be obtained from donor tissue by dissociation ofindividual cells from the connecting extracellular matrix of the tissue.Tissue from a particular neural region is removed from the brain using asterile procedure, and the cells are dissociated using any method knownin the art including treatment with enzymes such as trypsin, collagenaseand the like, or by using physical methods of dissociation such as witha blunt instrument. Dissociation of fetal cells can be carried out intissue culture medium, while a preferable medium for dissociation ofjuvenile and adult cells is low Ca²⁺ artificial cerebral spinal fluid(aCSF). Regular aCSF contains 124 mM NaCl, 5 mM KCl, 1.3 mM MgCl₂, 2 mMCaCl₂, 26 mM NaHCO₃, and 10 mM D-glucose. Low Ca²⁺ aCSF contains thesame ingredients except for MgCl₂ at a concentration of 3.2 mM and CaCl₂at a concentration of 0.1 mM. Dissociated cells are centrifuged at lowspeed, between 200 and 2000 rpm, usually between 400 and 800 rpm, andthen resuspended in culture medium. The neural cells can be cultured insuspension or on a fixed substrate. However, substrates tend to inducedifferentiation of the neural stem cell progeny. Thus, suspensioncultures are preferred if large numbers of undifferentiated neural stemcell progeny are desired. Cell suspensions are seeded in any receptaclecapable of sustaining cells, particularly culture flasks, culture platesor roller bottles, and more particularly in small culture flasks such as25 cm² culture flasks. Cells cultured in suspension are resuspended atapproximately 5×10⁴ to 2×10⁵ cells/ml, preferably 1×10⁵ cells/ml. Cellsplated on a fixed substrate are plated at approximately 2-3×10³cells/cm², preferably 2.5×10³ cells/cm².

[0084] The dissociated neural cells can be placed into any known culturemedium capable of supporting cell growth, including HEM, DMEM, RPMI,F-12, and the like, containing supplements which are required forcellular metabolism such as glutamine and other amino acids, vitamins,minerals and useful proteins such as transferrin and the like. Mediummay also contain antibiotics to prevent contamination with yeast,bacteria and fungi such as penicillin, streptomycin, gentamicin and thelike. In some cases, the medium may contain serum derived from bovine,equine, chicken and the like. However, a preferred embodiment forproliferation of neural stem cells is to use a defined, serum-freeculture medium, as serum tends to induce differentiation and containsunknown components (i.e. is undefined). A defined culture medium is alsopreferred if the cells are to be used for transplantation purposes. Aparticularly preferable culture medium is a defined culture mediumcomprising a mixture of DMEM, F12, and a defined hormone and saltmixture. This culture medium is referred to herein as “Complete Medium”and is described in detail in Example 3.

[0085] Conditions for culturing should be close to physiologicalconditions. The pH of the culture medium should be close tophysiological pH, preferably between pH 6-8, more preferably betweenabout pH 7 to 7.8, with pH 7.4 being most preferred. Physiologicaltemperatures range between about 30° C. to 40° C. Cells are preferablycultured at temperatures between about 32° C. to about 38° C., and morepreferably between about 35° C. to about 37° C.

[0086] The culture medium is supplemented with at least oneproliferation-inducing growth factor. As used herein, the term “growthfactor” refers to a protein, peptide or other molecule having a growth,proliferative, differentiative, or trophic effect on neural stem cellsand/or neural stem cell progeny. Growth factors which may be used forinducing proliferation include any trophic factor that allows neuralstem cells and precursor cells to proliferate, including any moleculewhich binds to a receptor on the surface of the cell to exert a trophic,or growth-inducing effect on the cell. Preferred proliferation-inducinggrowth factors include EGF, amphiregulin, acidic fibroblast growthfactor (aFGF or FGF-1), basic fibroblast growth factor (bFGF or FGF-2),transforming growth factor alpha (TGFσ), and combinations thereof.

[0087] Preferred proliferation-inducing growth factors include EGF andTGFα. A preferred combination of proliferation-inducing growth factorsis EGF or TGFα with FGF-1 or FGF-2. Growth factors are usually added tothe culture medium at concentrations ranging between about 1 fg/ml to 1mg/ml. Concentrations between about 1 to 100 ng/ml are usuallysufficient. Simple titration experiments can easily be performed todetermine the optimal concentration of a particular growth factor.

[0088] In addition to proliferation-inducing growth factors, othergrowth factors may be added to the culture medium that influenceproliferation and differentiation of the cells including NGF,platelet-derived growth factor (PDGF), thyrotropin releasing hormone(TRH), transforming growth factor betas (TGFβs), insulin-like growthfactor (IGF⁻¹) and the like.

[0089] Within 3-4 days in the presence of a proliferation-inducinggrowth factor, a multipotent neural stem cell begins to divide givingrise to a cluster of undifferentiated cells referred to herein as a“neurosphere”. The cells of a single neurosphere are clonal in naturebecause they are the progeny of a single neural stem cell. In thecontinued presence of a proliferation-inducing growth factor such as EGFor the like, precursor cells within the neurosphere continue to divideresulting in an increase in the size of the neurosphere and the numberof undifferentiated cells. The neurosphere is not immunoreactive forGFAP, neurofilament (NF), neuron-specific enolase (NSE) or myelin basicprotein (MBP). However, precursor cells within the neurosphere areimmunoreactive for nestin, an intermediate filament protein found inmany types of undifferentiated CNS cells. The nestin marker wascharacterized by Lehndahl et al., Cell 60:585-595 (1990). Antibodies areavailable to identify nestin, including the rat antibody referred to asRat401. The mature phenotypes associated with the differentiated celltypes that may be derived from the neural stem cell progeny arepredominantly negative for the nestin phenotype.

[0090] After about 4 to 5 days in the absence of a substrate, theproliferating neurospheres lift off the floor of the culture dish andtend to form the free-floating clusters characteristic of neurospheres.Floating neurospheres are depicted in FIG. 2d. It is possible to varythe culture conditions so that while the precursor cells still expressthe nestin phenotype, they do not form the characteristic neurospheres.The proliferating precursor cells of the neurosphere continue toproliferate in suspension. After about 3-10 days in vitro, and moreparticularly after about 6-7 days in vitro, the proliferatingneurospheres are fed every 2-7 days, preferably every 2-4 days by gentlecentrifugation and resuspension in Complete Medium containing a growthfactor.

[0091] The neurospheres of the suspension culture can be easily passagedto reinitiate proliferation. After 6-7 days in vitro, the culture flasksare shaken well and the neurospheres allowed to settle on the bottomcorner of the flask. The neurospheres are then transferred to a 50 mlcentrifuge tube and centrifuged at low speed. The medium is aspirated,and the neurospheres are resuspended in a small amount of CompleteMedium. Individual cells in the neurospheres can be separated byphysical dissociation of the neurospheres with a blunt instrument, forexample, by triturating the neurospheres with a pipette, especially afire polished pasteur pipette, to form a single cell suspension ofneural stem cell progeny. The cells are then counted and replated at thedesired density to reinitiate proliferation. Single cells from thedissociated neurospheres are suspended in Complete Medium containinggrowth factor, and a percentage of these cells proliferate and form newneurospheres largely composed of undifferentiated cells. This procedurecan be repeated weekly to result in a logarithmic increase in the numberof viable cells at each passage. The procedure is continued until thedesired number of precursor cells is obtained.

[0092] The number neural stem cell progeny proliferated in vitro fromthe mammalian CNS can be increased dramatically by injecting a growthfactor or combination of growth factors, for example EGF, FGF, or EGFand FGF together, into the ventricles of the donor in vivo using the invivo proliferation methods described in more detail below. As detailedin Example 31 below, 6 days after infusion of EGF into the lateralventricle of a mouse forebrain, the walls of the ventricle were removedand the stem cells harvested. Infusion of EGF into the lateral ventricleincreased the efficiency of the yield of stem cells that proliferated toform neurospheres.

[0093] This ability to enhance the proliferation of neural stem cellsshould prove invaluable when stem cells are to be harvested for latertransplantation back into a patient, thereby making the initialsurgery 1) less traumatic because less tissue would have to be removedand 2) more efficient because a greater yield of stem cells per surgerywould proliferate in vitro.

[0094] Additionally, the patient's stem cells, once they haveproliferated in vitro, could also be genetically modified in vitro usingthe techniques described below. The in vitro genetic modification may bemore desirable in certain circumstances than in vivo geneticmodification techniques when more control over the infection with thegenetic material is required.

[0095] Neural stem cell progeny can be cryopreserved until they areneeded by any method known in the art The cells can be suspended in anisotonic solution, preferably a cell culture medium, containing aparticular cryopreservant. Such cryopreservants include dimethylsulfoxide (DMSO), glycerol and the like. These cryopreservants are usedat a concentration of 5-15%, preferably 8-10%. Cells are frozengradually to a temperature of −10° C. to −150° C., preferably −20° C. to−100° C., and more preferably −70° C. to −80° C.

[0096] Differentiation of Neural Stem Cell Progeny

[0097] Differentiation of the cells can be induced by any method knownin the art which activates the cascade of biological events which leadto growth, which include the liberation of inositol triphosphate andintracellular Ca²⁺, liberation of diacyl glycerol and the activation ofprotein kinase C and other cellular kinases, and the like. Treatmentwith phorbol esters, differentiation-inducing growth factors and otherchemical signals can induce differentiation. Differentiation can also beinduced by plating the cells on a fixed substrate such as flasks,plates, or coverslips coated with an ionically charged surface such aspoly-L-lysine and poly-L-ornithine and the like.

[0098] Other substrates may be used to induce differentiation such ascollagen, fibronectin, laminin, matrigel, and the like. Differentiationcan also be induced by leaving the cells in suspension in the presenceof a proliferation-inducing growth factor, without reinitiation ofproliferation (i.e. without dissociating the neurospheres).

[0099] A preferred method for inducing differentiation of the neuralstem cell progeny comprises culturing the cells on a fixed substrate ina culture medium that is free of the proliferation-inducing growthfactor. After removal of the proliferation-inducing growth factor, thecells adhere to the substrate (e.g. poly-ornithine-treated plastic orglass), flatten, and begin to differentiate into neurons and glialcells. At this stage the culture medium may contain serum such as0.5-1.0% fetal bovine serum (FBS). However, for certain uses, if definedconditions are required, serum would not be used. Within 2-3 days, mostor all of the neural stem cell progeny begin to lose immunoreactivityfor nestin and begin to express antigens specific for neurons,astrocytes or oligodendrocytes as determined by immunocytochemistrytechniques well known in the art.

[0100] Immunocytochemistry (e.g. dual-label immunofluorescence andimmunoperoxidase methods) utilizes antibodies that detect cell proteinsto distinguish the cellular characteristics or phenotypic properties ofneurons from astrocytes and oligodendrocytes. In particular, cellularmarkers for neurons include NSE, NF, β-tub, MAP-2; and for glia, GFAP(an identifier of astrocytes), galactocerebroside (GalC) (a myelinglycolipid identifier of oligodendrocytes), and the like.

[0101] Immunocytochemistry can also be used to detect the expression ofneurotransmitters, or in some cases the expression of enzymesresponsible for neurotransmitter synthesis. For the identification ofneurons, antibodies can be used that detect the presence ofacetylcholine (ACh), dopamine, epinephrine, norepinephrine, histamine,serotonin or 5-hydroxytryptamine (5-HT), neuropeptides such as substanceP, adrenocorticotrophic hormone, vasopressin or anti-diuretic hormone,oxytocin, somatostatin, angiotensin II, neurotensin, and bombesin,hypothalamic releasing hormones such as TRH and luteinizing releasinghormone, gastrointestinal peptides such as vasoactive intestinal peptide(VIP) and cholecystokinin (CCK) and CCK-like peptide, opioid peptidessuch as endorphins like β-endorphin and enkephalins such as met- andleu-enkephalin, prostaglandins, amino acids such as GABA, glycine,glutamate, cysteine, taurine and aspartate and dipeptides such ascarnosine. Antibodies to neurotransmitter-synthesizing enzymes can alsobe used such as glutamic acid decarboxylase (GAD) which is involved inthe synthesis of GABA, choline acetyltransferase (ChAT) for AChsynthesis, dopa decarboxylase (DDC) for dopamine, dopamine-β-hydroxylase(DBH) for norepinephrine, and amino acid decarboxylase for 5-HT.Antibodies to enzymes that are involved in the deactivation ofneurotransmitters may also be useful such as acetyl cholinesterase(AChE) which deactivates ACh. Antibodies to enzymes involved in thereuptake of neurotransmitters into neuronal terminals such as monoamineoxidase and catechol-o-methyl transferase for dopamine, for 5-HT, andGABA transferase for GABA may also identify neurons. Other markers forneurons include antibodies to neurotransmitter receptors such as theAChE nicotinic and muscarinic receptors, adrenergic receptors α¹, α₂, β¹and α₂, the dopamine receptor and the like. Cells that contain a highlevel of melanin, such as those found in the substantia nigra, could beidentified using an antibody to melanin.

[0102] In situ hybridization histochemistry can also be performed, usingcDNA or RNA probes specific for the peptide neurotransmitter or theneurotransmitter synthesizing enzyme mRNAs. These techniques can becombined with immunocytochemical methods to enhance the identificationof specific phenotypes. If necessary, the antibodies and molecularprobes discussed above can be applied to Western and Northern blotprocedures respectively to aid in cell identification.

[0103] A preferred method for the identification of neurons usesimmunocytochemistry to detect immunoreactivity for NSE, NF, NeuN, andthe neuron specific protein, tau-1. Because these markers are highlyreliable, they will continue to be useful for the primary identificationof neurons, however neurons can also be identified based on theirspecific neurotransmitter phenotype as previously described.

[0104] Type I astrocytes, which are differentiated glial cells that havea flat, protoplasmic/fibroblast-like morphology, are preferablyidentified by their immunoreactivity for GFAP but not A2B5. Type IIastrocytes, which are differentiated glial cells that display a stellateprocess-bearing morphology, are preferably identified usingimmunocytochemistry by their phenotype GFAP(+), A2B5(+) phenotype.

[0105] Cells that do not express intermediate filaments specific forneurons or for astrocytes, begin to express markers specific foroligodendrocytes in a correct temporal fashion. That is, the cells firstbecome immunoreactive for O4, galactocerebroside (GalC, a myelinglycolipid) and finally, MBP. These cells also possess a characteristicoligodendrocyte morphology.

[0106] The present invention provides a method of influencing therelative proportion of these differentiated cell types by the additionof exogenous growth factors during the differentiation stage of theprecursor cells. By using dual-label immunofluorescence andimmunoperoxidase methods with various neuronal- and glial-specificantibodies, the effect of the exogenous growth factors on thedifferentiating cells can be determined.

[0107] The biological effects of growth and trophic factors aregenerally mediated through binding to cell surface receptors. Thereceptors for a number of these factors have been identified andantibodies and molecular probes for specific receptors are available.Neural stem cells can be analyzed for the presence of growth factorreceptors at all stages of differentiation. In many cases, theidentification of a particular receptor will define the strategy to usein further differentiating the cells along specific developmentalpathways with the addition of exogenous growth or trophic factors.

[0108] Exogenous growth factors can be added alone or in variouscombinations. They can also be added in a temporal sequence (i.e.exposure to a first growth factor influences the expression of a secondgrowth factor receptor, Neuron 4:189-201 (1990). Among the growthfactors and other molecules that can be used to influence thedifferentiation of precursor cells in vitro are FGF-1, FGF-2, ciliaryneurotrophic factor (CNTF), NGF, BDNF, neurotrophin 3, neurotrophin 4,interleulins, leukemia inhibitory factor (LIF), cyclic adenosinemonophosphate, forskolin, tetanus toxin, high levels of potassium,amphiregulin, TGF-α, TGF-β, insulin-like growth factors, dexamethasone(glucocorticoid hormone), isobutyl 3-methylxanthine, somatostatin,growth hormone, retinoic acid, and PDGF. These and other growth factorsand molecules will find use in the present invention.

[0109] Genetic Modification of Neural Stem Cell Progeny

[0110] Although the precursor cells are non-transformed primary cells,they possess features of a continuous cell line. In the undifferentiatedstate, in the presence of a proliferation-inducing growth factor such asEGF, the cells continuously divide and are therefore excellent targetsfor genetic modification. The term “genetic modification” as used hereinrefers to the stable or transient alteration of the genotype of aprecursor cell by intentional introduction of exogenous DNA. DNA may besynthetic, or naturally derived, and may contain genes, portions ofgenes, or other useful DNA sequences. The term “genetic modification” asused herein is not meant to include naturally occurring alterations suchas that which occurs through natural viral activity, natural geneticrecombination, or the like.

[0111] Exogenous DNA may be introduced to a precursor cell by viralvectors (retrovirus, modified herpes viral, herpes-viral, adenovirus,adeno-associated virus, and the like) or direct DNA transfection(lipofection, calcium phosphate transfection, DEAE-dextran,electroporation, and the like). The genetically modified cells of thepresent invention possess the added advantage of having the capacity tofully differentiate to produce neurons or macroglial cells in areproducible fashion using a number of differentiation protocols.

[0112] In another embodiment, the precursor cells are derived fromtransgenic animals, and thus are in a sense already geneticallymodified. There are several methods presently used for generatingtransgenic animals. The technique used most often is directmicroinjection of DNA into single-celled fertilized eggs. Othertechniques include retroviral-mediated transfer, or gene transfer inembryonic stem cells. These techniques and others are detailed by Hoganet al. in Manipulating the Mouse Embryo, A Laboratory Manual (ColdSpring Harbor Laboratory Ed., 1986). Use of these transgenic animals hascertain advantages including the fact that there is no need to transfecthealthy neurospheres. Precursor cells derived from transgenic animalswill exhibit stable gene expression. Using transgenic animals, it ispossible to breed in new genetic combinations. The transgenic animal mayhave integrated into its genome any useful gene that is expressed byneural cells. Examples of useful DNA are given below in the discussionof genetically modifying precursor cells.

[0113] A significant challenge for cellular transplantation in the CNSis the identification of the donor cells after implantation within thehost. A number of strategies have been employed to mark donor cells,including tritiated labels, fluorescent dyes, dextrans, and viralvectors carrying reporter genes. However, these methods suffer frominherent problems of toxicity, stability, or dilution over the longterm. The use of neural cells derived from transgenic animals mayprovide an improved means by which identification of transplanted neuralcells can be achieved. A transgenic marking system provides a morestable and efficient method for cell labeling. In this system, promoterelements, for example for GFAP and MBP, can direct the expression of theE. coli B-galactosidase reporter gene in transgenic mice. In thesesystems, cell-specific expression of the reporter gene occurs inastrocytes (GFAP-lacZ) and in oligodendrocytes (MBP-lacZ) in adevelopmentally-regulated manner. The Rosa26 transgenic mice, describedin Example 45, is one example of a transgenic marking system in whichall cells ubiquitously express β-galactosidase.

[0114] Once propagated, the neurosphere cells are mechanicallydissociated into a single cell suspension and plated on petri dishes ina medium where they are allowed to attach overnight. The precursor cellsare then genetically modified. If the precursor cells are generated fromtransgenic animals, then they may or may not be subjected to furthergenetic modification, depending upon the properties desired of thecells. Any useful genetic modification of the cells is within the scopeof the present invention. For example, precursor cells may be modifiedto produce or increase production of a biologically active substancesuch as a neurotransmitter or growth factor or the like. The geneticmodification is performed either by infection with recombinantretroviruses or transfection using methods known in the art (seeManiatis et al., in Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory, N.Y. (1982)). Briefly, the chimeric gene constructswill contain viral, for example retroviral long terminal repeat (LTR),simian virus 40 (SV40), cytomegalovirus (CMV); or mammaliancell-specific promoters such as tyrosine hydroxylase (TH, a marker fordopamine cells), DBH, phenylethanolamine N-methyltransferase (PNMT),ChAT, GFAP, NSE, the NF proteins (NF-L, NF-M, NF-H, and the like) thatdirect the expression of the structural genes encoding the desiredprotein. In addition, the vectors will include a drug selection marker,such as the E. coli aminoglycoside phosphotransferase gene, which whencoinfected with the experimental gene confers resistance to geneticin(G418), a protein synthesis inhibitor.

[0115] When the genetic modification is for the production of abiologically active substance, the substance will generally be one thatis useful for the treatment of a given CNS disorder. For example, it maybe desired to genetically modify cells so they secrete a certain growthfactor product. As used herein, the term “growth factor product” refersto a protein, peptide, mitogen, or other molecule having a growth,proliferative, differentiative, or trophic effect. Growth factorproducts useful in the treatment of CNS disorders include, but are notlimited to, NGF, BDNF, the neurotrophins (NT-3, NT-4/NT-5), CNTF,amphiregulin, FGF-1, FGF-2, EGF, TGFα, TGFβs, PDGF, IGFs, and theinterleukins.

[0116] Cells can also be modified to express a certain growth factorreceptor (r) including, but not limited to, p75 low affinity NGFr,CNTFr, the trk family of neurotrophin receptors (trk, trkB, trkC), EGFr,FGFr, and amphiregulin receptors. Cells can be engineered to producevarious neurotransmitters or their receptors such as serotonin, L-dopa,dopamine, norepinephrine, epinephrine, tachykinin, substance-P,endorphin, enkephalin, histamine, N-methyl D-aspartate, glycine,glutamate, GABA, ACh, and the like. Useful neurotransmitter-synthesizinggenes include TH, DDC, DBH, PNMT, GAD, tryptophan hydroxylase, ChAT, andhistidine decarboxylase. Genes that encode for various neuropeptides,which may prove useful in the treatment of CNS disorders, includesubstance-P, neuropeptide-Y, enkephalin, vasopressin, VIP, glucagon,bombesin, CCK, somatostatin, calcitonin gene-related peptide, and thelike.

[0117] After successfully transfected/infected cells are selected theycan be cloned using limiting dilution in 96 multi-well plates andassayed for the presence of the desired biologically active substance.Clones that express high levels of the desired substance are grown andtheir numbers expanded in T-flasks. The specific cell line can then becyropreserved. Multiple clones of genetically modified precursor cellswill be obtained. Some may give rise preferentially to neuronal cells,and some to glial cells.

[0118] The genetically modified precursor cells can be implanted forcell/gene therapy into the CNS of a recipient in need of thebiologically active molecule produced by the genetically modified cellsTransplantation techniques are detailed below.

[0119] Alternatively, the genetically modified precursor cells can besubjected to various differentiation protocols in vitro prior toimplantation. For example, genetically modified precursor cells may beremoved from the culture medium which allows proliferation anddifferentiated using any of the protocols described above. The protocolused will depend upon the type of genetically modified cell desired.Once the cells have differentiated, they are again assayed forexpression of the desired protein. Cells having the desired phenotypecan be isolated and implanted into recipients in need of the protein orbiologically active molecule that is expressed by the geneticallymodified cell.

[0120] Transplantation of Neural Stem Cell Progeny Alleviate Disordersof the CNS in Animal Models Caused by Disease or Injury

[0121] It is well recognized in the art that transplantation of tissueinto the CNS offers the potential for treatment of neurodegenerativedisorders and CNS damage due to injury (review: Lindvall, (1991) Tinsvol. 14(8): 376-383). Transplantation of new cells into the damaged CNShas the potential to repair damaged circuitries and provideneurotransmitters thereby restoring neurological function. However, theabsence of suitable cells for transplantation purposes has prevented thefull potential of this procedure from being met. “Suitable” cells arecells that meet the following criteria: 1) can be obtained in largenumbers; 2) can be proliferated in vitro to allow insertion of geneticmaterial, if necessary; 3) capable of surviving indefinitely but stopgrowing after transplantation to the brain; 4) are non-immunogenic,preferably obtained from a patient's own tissue; 5) are able to formnormal neural connections and respond to neural physiological signals(Bjorklund (1991) TINS Vol. 14(8): 319-322). The progeny of multipotentneural stem cells obtainable from embryonic or adult CNS tissue, whichare able to divide indefinitely when maintained in vitro using theculture conditions described herein, meet all of the desirablerequirements of cells suitable for neural transplantation purposes andare a particularly suitable cell line as the cells have not beenimmortalized and are not of tumorigenic origin. The use of multipotentneural stem cells in the treatment of neurological disorders and CNSdamage can be demonstrated by the use of animal models.

[0122] The neural stem cell progeny can be administered to any animalwith abnormal neurological or neurodegenerative symptoms obtained in anymanner, including those obtained as a result of mechanical, chemical, orelectrolytic lesions, as a result of experimental aspiration of neuralareas, or as a result of aging processes. Particularly preferablelesions in non-human animal models are obtained with 6-hydroxy-dopamine(6-OHDA), 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP), ibotenicacid and the like.

[0123] The instant invention allows the use of precursor cells preparedfrom donor tissue which is xenogeneic to the host. Since the CNS is asomewhat immunoprivileged site, the immune response is significantlyless to xenografts, than elsewhere in the body. In general, however, inorder for xenografts to be successful it is preferred that some methodof reducing or eliminating the immune response to the implanted tissuebe employed. Thus recipients will often be immunosuppressed, eitherthrough the use of immunosuppressive drugs such as cyclosporin, orthrough local immunosuppression strategies employing locally appliedimmunosuppressants Local immunosuppression is disclosed by Gruber,Transplantation 54:1-11 (1992). Rossini, U.S. Pat. No. 5,026,365,discloses encapsulation methods suitable for local immunosuppression.

[0124] As an alternative to employing immunosuppression techniques,methods of gene replacement or knockout using homologous recombinationin embryonic stem cells, taught by Smithies et al. Nature, 317:230-234(1985), and extended to gene replacement or knockout in cell lines (H.Zheng 35 al., PNAS, 88:8067-8071 (1991)), can be applied to precursorcells for the ablation of major histocompatibility complex (MHC) genes.Precursor cells lacking MHC expression would allow for the grafting ofenriched neural cell populations across allogeneic, and perhaps evenxenogeneic, histocompatibility barriers without the need toimmunosuppress the recipient. General reviews and citations for the useof recombinant methods to reduce antigenicity of donor cells are alsodisclosed by Gruber (supra). Exemplary approaches to the reduction ofimmunogenicity of transplants by surface modification are disclosed byFaustman WO 92/04033 (1992). Alternatively the immunogenicity of thegraft may be reduced by preparing precursor cells from a transgenicanimal that has altered or deleted MHC antigens.

[0125] Grafting of precursor cells prepared from tissue which isallogeneic to that of the recipient will most often employ tissue typingin an effort to most closely match the histocompatibility type of therecipient. Donor cell age as well as age of the recipient have beendemonstrated to be important factors in improving the probability ofneuronal graft survival. The efficiency of grafting is reduced withincreased age of donor cells. Furthermore, grafts are more readilyaccepted by younger recipients compared to older recipients. These twofactors are likely to be as important for glial graft survival as theyare for neuronal graft survival.

[0126] In some instances, it may be possible to prepare neural stem cellprogeny from the recipient's own nervous system (e.g. in the case oftumor removal biopsies etc,). In such instances the neural stem cellprogeny may be generated from dissociated tissue and proliferated invitro using the methods described above. Upon suitable expansion of cellnumbers, the precursor cells may be harvested, genetically modified ifnecessary, and readied for direct injection into the recipient's CNS.

[0127] Transplantation can be done bilaterally, or, in the case of apatient suffering from Parkinson's Disease, contralateral to the mostaffected side. Surgery is performed in a manner in which particularbrain regions may be located, such as in relation to skull sutures,particularly with a stereotaxic guide. Cells are delivered throughoutany affected neural area, in particular to the basal ganglia, andpreferably to the caudate and putamen, the nucleus basalis or thesubstantia nigra. Cells are administered to the particular region usingany method which maintains the integrity of surrounding areas of thebrain, preferably by injection cannula. Injection methods exemplified bythose used by Duncan et al. J.Neurocytology, 17:351-361 (1988), andscaled up and modified for use in humans are preferred. Methods taughtby Gage et al., supra, for the injection of cell suspensions such asfibroblasts into the CNS may also be employed for injection of neuralprecursor cells. Additional approaches and methods may be found inNeural Grafting in the Mammalian CNS, Bjorklund and Stenevi, eds.,(1985).

[0128] Although solid tissue fragments and cell suspensions of neuraltissue are immunogenic as a whole, it could be possible that individualcell types within the graft are themselves immunogenic to a lesserdegree. For example, Bartlett et al. (Prog. Brain Res. 82: 153-160(1990)) have abrogated neural allograft rejection by pre-selecting asubpopulation of embryonic neuroepithelial cells for grafting by the useof immunobead separation on the basis of MHC expression. Thus, anotherapproach is provided to reduce the chances of allo and xenograftrejection by the recipient without the use of immunosuppressiontechniques.

[0129] Neural stem cell progeny when administered to the particularneural region preferably form a neural graft, wherein the neuronal cellsform normal neuronal or synaptic connections with neighboring neurons,and maintain contact with transplanted or existing glial cells which mayform myelin sheaths around the neurons' axons, and provide a trophicinfluence for the neurons. As these transplanted cells form connections,they re-establish the neuronal networks which have been damaged due todisease and aging.

[0130] Survival of the graft in the living host can be examined usingvarious non-invasive scans such as computerized axial tomography (CATscan or CT scan), nuclear magnetic resonance or magnetic resonanceimaging (NMR or MRI) or more preferably positron emission tomography(PET) scans. Post-mortem examination of graft survival can be done byremoving the neural tissue, and examining the affected regionmacroscopically, or more preferably using microscopy. Cells can bestained with any stains visible under light or electron microscopicconditions, more particularly with stains which are specific for neuronsand glib Particularly useful are monoclonal antibodies which identifyneuronal cell surface markers such as the M6 antibody which identifiesmouse neurons. Most preferable are antibodies which identify anyneurotransmitters, particularly those directed to GABA, TH, ChAT, andsubstance P, and to enzymes involved in the synthesis ofneurotransmitters, in particular, GAD. Transplanted cells can also beidentified by prior incorporation of tracer dyes such as rhodamine- orfluorescein-labelled microspheres, fast blue, bisbenzamide orretrovirally introduced histochemical markers such as the lac Z genewhich produces beta galactosidase.

[0131] Functional integration of the graft into the host's neural tissuecan be assessed by examining the effectiveness of grafts on restoringvarious functions, including but not limited to tests for endocrine,motor, cognitive and sensory functions. Motor tests which can be usedinclude those which quantitate rotational movement away from thedegenerated side of the brain, and those which quantitate slowness ofmovement, balance, coordination, akinesia or lack of movement, rigidityand tremors. Cognitive tests include various tests of ability to performeveryday tasks, as well as various memory tests, including mazeperformance.

[0132] Neural stem cell progeny can be produced and transplanted usingthe above procedures to treat demyelination diseases. Humandemyelinating diseases for which the cells of the present invention mayprovide treatment include disseminated perivenous encephalomyelitis, MS(Charcot and Marburg types), neuromyelitis optica, concentric sclerosis,acute, disseminated encephalomyelitides, post encephalomyelitis,postvaccinal encephalomyelitis, acute hemorrhagic leukoencephalopathy,progressive multifocal leukoencephalopathy, idiopathic polyneuritis,diphtheric neuropathy, Pelizaeus-Merzbacher disease, neuromyelitisoptica, diffuse cerebral sclerosis, central pontine myelinosis,spongiform leukodystrophy, and leukodystrophy (Alexander type).

[0133] Areas of demyelination in humans is generally associated withplaque like structures. Plaques can be visualized by magnetic resonanceimaging. Accessible plaques are the target area for injection of neuralstem cell progeny. Standard stereotactic neurosurgical methods are usedto inject cell suspensions both into the brain and spinal cord.Generally, the cells can be obtained from any of the sources discussedabove. However, in the case of demyelinating diseases with a geneticbasis directly affecting the ability of the myelin forming cell tomyelinate axons, allogeneic tissue would be a preferred source of thecells as autologous tissue (i.e. the recipient's cells) would generallynot be useful unless the cells have been modified in some way to insurethe lesion will not continue (e.g. genetically modifying the cells tocure the demyelination lesion).

[0134] Oligodendrocytes derived from neural stem cell progenyproliferated and differentiated in vitro may be injected intodemyelinated target areas in the recipient. Appropriate amounts of typeI astrocytes may also be injected. Type I astrocytes are known tosecrete PDGF which promotes both migration and cell division ofoligodendrocytes. [Nobel et al., Nature 333:560-652 (1988); Richardsonet al., Cell, 53:309-319 (1988)].

[0135] A preferred treatment of demyelination disease usesundifferentiated neural stem cell progeny. Neurospheres grown in thepresence of a proliferation-inducing growth factor such as EGF can bedissociated to obtain individual precursor cells which are then placedin injection medium and injected directly into the demyelinated targetregion. The cells differentiate in vivo. Astrocytes can promoteremyelination in various paradigms. Therefore, in instances whereoligodendrocyte proliferation is important, the ability of precursorcells to give rise to type I astrocytes may be useful. In othersituations, PDGF may be applied topically during the transplantation aswell as with repeated doses to the implant site thereafter.

[0136] The injection of neural stem cell progeny in remyelinationtherapy provides, amongst other types of cells, a source of immaturetype I astrocytes at the implant site. This is a significant featurebecause immature astrocytes (as opposed to mature astrocytes) have anumber of specific characteristics that make them particularly suitedfor remyelination therapy. First, immature, as opposed to mature, type Iastrocytes are known to migrate away from the implant site [Lindsay et.al, Neurosci. 12:513-530 (1984)] when implanted into a mature recipientand become associated with blood vessels in the recipient's CNS [Silveret al., WO 91/06631 (1991)]. This is at least partially due to the factthat immature astrocytes are intrinsically more motile than matureastrocytes. [Duffy et al., Exp Cell Res. 139:145-157 (1982), Table VII].Type I astrocytes differentiating at or near the precursor cell implantsite should have maximal motility and thereby optimize the opportunityfor oligodendrocyte growth and division at sites distant from theimplant. The localization of the astrocytes near blood vessels is alsosignificant from a therapeutic standpoint since (at least in MS) mostplaques have a close anatomical relationship with one or more veins.

[0137] Another characteristic of immature astrocytes that makes themparticularly suited for remyelination therapy is that they undergo alesser degree of cell death than mature type I astrocytes. (Silver etal., supra)

[0138] Any suitable method for the implantation of precursor cells nearto the demyelinated targets may be used so that the cells can becomeassociated with the demyelinated axons. Glial cells are motile and areknown to migrate to, along, and across their neuronal targets therebyallowing the spacing of injections. Remyelination by the injection ofprecursor cells is a useful therapeutic in a wide range of demyelinatingconditions. It should also be borne in mind that in some circumstancesremyelination by precursor cells will not result in permanentremyelination, and repeated injections will be required. Suchtherapeutic approaches offer advantage over leaving the conditionuntreated and may spare the recipient's life.

[0139] In Vivo Proliferation, Differentiation, and Genetic Modificationof Neural Stem Cell Progeny

[0140] Neural stem cells and their progeny can be induced to proliferateand differentiate in vivo by administering to the host, any growthfactor(s) or pharmaceutical composition that will induce proliferationand differentiation of the cells. These growth factors include anygrowth factor known in the art, including the growth factors describedabove for in vitro proliferation and differentiation. Pharmaceuticalcompositions include any substance that blocks the inhibitory influenceand/or stimulates neural stem cells and stem cell progeny to proliferateand ultimately differentiate. Thus, the techniques described above toproliferate, differentiate, and genetically modify neural stem cells invitro can be adapted to in vivo techniques, to achieve similar results.Such in vivo manipulation and modification of these cells allows cellslost, due to injury or disease, to be endogenously replaced, thusobviating the need for transplanting foreign cells into a patient.Additionally, the cells can be modified or genetically engineered invivo so that they express various biological agents useful in thetreatment of neurological disorders.

[0141] Administration of growth factors can be done by any method,including injection cannula, transfection of cells with growthhormone-expressing vectors, injection, timed-release apparati which canadminister substances at the desired site, and the like. Pharmaceuticalcompositions can be administered by any method, including injectioncannula, injection, oral administration, timed-release apparati and thelike. The neural stem cells can be induced to proliferate anddifferentiate in vivo by induction with particular growth factors orpharmaceutical compositions which will induce their proliferation anddifferentiation. Therefore, this latter method circumvents the problemsassociated with transplantation and immune reactions to foreign cells.Any growth factor can be used, particularly EGF, TGFα, FGF-1, FGF-2 andNGF.

[0142] Growth factors can be administered in any manner known in the artin which the factors may either pass through or by-pass the blood-brainbarrier. Methods for allowing factors to pass through the blood-brainbarrier include minimizing the size of the factor, or providinghydrophobic factors which may pass through more easily.

[0143] The fact that neural stem cells are located in the tissues liningventricles of mature brains offers several advantages for themodification and manipulation of these cells in vivo and the ultimatetreatment of various neurological diseases, disorders, and injury thataffect different regions of the CNS. Therapy for these can be tailoredaccordingly so that stem cells surrounding ventricles near the affectedregion would be manipulated or modified in vivo using the methodsdescribed herein. The ventricular system is found in nearly all brainregions and thus allows easier access to the affected areas. If onewants to modify the stem cells in vivo by exposing them to a compositioncomprising a growth factor or a viral vector, it is relatively easy toimplant a device that administers the composition to the ventricle andthus, to the neural stem cells. For example, a cannula attached to anosmotic pump may be used to deliver the composition. Alternatively, thecomposition may be injected directly into the ventricles. The neuralstem cell progeny can migrate into regions that have been damaged as aresult of injury or disease. Furthermore, the close proximity of theventricles to many brain regions would allow for the diffusion of asecreted neurological agent by the stem cells or their progeny.

[0144] For treatment of Huntington's Disease, Alzheimer's Disease,Parkinson's Disease, and other neurological disorders affectingprimarily the forebrain, growth factors or other neurological agentswould be delivered to the ventricles of the forebrain to affect in vivomodification or manipulation of the stem cells. For example, Parlinson'sDisease is the result of low levels of dopamine in the brain,particularly the striatum. It would be advantageous to induce apatient's own quiescent stem cells to begin to divide in vivo and toinduce the progeny of these cells to differentiate into dopaminergiccells in the affected region of the striatum, thus locally raising thelevels of dopamine.

[0145] Normally the cell bodies of dopaminergic neurons are located inthe substantia nigra and adjacent regions of the mesencephalon, with theaxons projecting to the striatum. Prior art methods for treatingParkinson's disease usually involves the use of the drug L-Dopa, toraise dopamine levels in the striatum. However, there are disadvantageswith this treatment including drug tolerance and side effects. Also,embryonic tissues that produce dopamine have been transplanted into thestriatum of human Parkinsonian patients with reasonable success.However, the use of large quantities of fetal human tissue required forthis procedure raises serious ethical concerns and practical issues.

[0146] The methods and compositions of the present invention provide analternative to the use of drugs and the controversial use of largequantities of embryonic tissue for treatment of Parkinson's disease.Dopamine cells can be generated in the striatum by the administration ofa composition comprising growth factors to the lateral ventricle. Aparticularly preferred composition comprises a combination of EGF,FGF-2, and heparan sulphate. The composition preferably also comprisesserum. After administration of this composition, there is a significantincrease in the transcription of messenger RNA (mRNA) for TH in thesubventricular region of the striatum, an area which normally does notcontain dopaminergic cell bodies. These methods and results aredescribed in detail in Example 34. As detailed in Example 35, the use ofdual labeling tissue to show the distribution of BrdU+ and TH+ cellsindicates that, in response to the in vivo administration of growthfactors, TH+ cell bodies occur in striatal tissue. Many of these newlygenerated TH+ cells are also BrdU+.

[0147] For the treatment of MS and other demyelinating orhypomyelinating disorders, and for the treatment of Amyotrophic LateralSclerosis or other motor neuron diseases, growth factors or otherneurological agents would be delivered to the central canal.

[0148] In addition to treating CNS tissue immediately surrounding aventricle, a viral vector, DNA, growth factor, or other neurologicalagent can be easily administered to the lumbar cistern for circulationthroughout the CNS.

[0149] Under normal conditions subependymal precursors do notdifferentiate or migrate, rather, their fate appears to be cell deathafter an undefined number of cell divisions (Morshead and Van der Kooy,supra). This explanation is also supported by PCR evidence, as describedabove. Injection of growth factors into the lateral ventricle altersthis fate. As described in more detail in Example 27 below, retroviruseswere injected into the lateral ventricles for six consecutive days.Implanting cannulae attached to EGF-filled osmotic pumps into thelateral ventricles on the same day as (and 1 or 6 days following)retrovirus injection results in an increase in the total number ofRV-β-gal labelled cells 6 days later (from an average of 20 cells/brainto 150 cells/brain).

[0150] It is known from the PCR experiments described above that 6 daysfollowing retroviral injection no cells exist that contain non-expressedretroviral DNA. Thus these results indicate that the EGF-inducedincrease in β-gal positive cell number is due to the expansion of theclone size of the retrovirally labelled constitutively proliferativepopulation. It is also possible that part of this increase is due to theactivation by EGF of a relatively quiescent stem cell.

[0151] Interestingly, this expansion of the number of β-gal labelledcells is accompanied by the migration of these cells away from thesubependymal medially, laterally, rostrally, and caudally withsubsequent differentiation. Thus, infusion of EGF or similar growthfactors induces the proliferation, migration and differentiation ofneural stem cells and progenitor cells in vivo, and can be usedtherapeutically to replace neural cells lost due to injury or disease.In a preferred embodiment EGF and FGF are administered together orsequentially.

[0152] The normal fate of the constitutively proliferating cellpopulation (i.e. cell death) can be altered by administering Bcl-2 orgenetically modifying the cells with the bcl-2 gene. The gene product isknown to prevent programmed cell death (apoptosis) in a variety of celltypes. Similar to the EGF experiments, a clonal expansion of theconstitutively proliferating cell population is achieved followinginfection with bcl-2.

[0153] Other ways of passing the blood-brain barrier include in vivotransfection of neural stem cells and stem cell progeny with expressionvectors containing genes that code for growth factors, so that the cellsthemselves produce the factor. Any useful genetic modification of thecells is within the scope of the present invention. For example, inaddition to genetic modification of the cells to express growth factors,the cells may be modified to express other types of neurological agentssuch as neurotransmitters. Preferably, the genetic modification isperformed either by infection of the cells lining ventricular regionswith recombinant retroviruses or transfection using methods known in theart including CaPO₄ transfection, DEAE-dextran transfection, polybrenetransfection, by protoplast fusion, electroporation, lipofection, andthe like [see Maniatis et al., supra]. Any method of geneticmodification, now known or later developed can be used. With direct DNAtransfection, cells could be modified by particle bombardment, receptormediated delivery, and cationic liposomes. When chimeric gene constructsare used, they generally will contain viral, for example retroviral longterminal repeat (LTR), simian virus 40 (SV40), cytomegalovirus (CMV); ormammalian cell-specific promoters such as those for TH, DBH,phenylethanolamine N-methyltransferase, ChAT, GFAP, NSE, the NF proteins(NF-L, NF-M, NF-H, and the like) that direct the expression of thestructural genes encoding the desired protein

[0154] If a retroviral construct is to be used to genetically modifynormally quiescent stem cells, then it is preferable to induce theproliferation of these cells using the methods described herein. Forexample, an osmotic infusion pump could be used to deliver growthfactors to the central canal several days prior to infection with theretrovirus. This assures that there will be actively dividing neuralstem cells which are susceptible to infection with the retrovirus.

[0155] When the genetic modification is for the production of abiologically active substance, the substance will generally be one thatis useful for the treatment of a given CNS disorder. For example, it maybe desired to genetically modify cells so they secrete a certain growthfactor product. Growth factor products useful in the treatment of CNSdisorders are listed above. Cells can also be modified in vivo toexpress a growth factor receptors, neurotransmitters or their receptors,neurotransmitter-synthesizing genes, neuropeptides, and the like, asdiscussed above.

[0156] Any expression vector known in the art can be used to express thegrowth factor, as long as it has a promoter which is active in the cell,and appropriate termination and polyadenylation signals. Theseexpression vectors include recombinant vaccinia virus vectors includingpSCll, or vectors derived various viruses such as from Simian Virus 40(SV40, i.e. pSV2-dhfr, pSV2neo, pko-neo, pSV2gpt, pSVT7 and pBABY), fromRous Sarcoma Virus (RSV, i.e. pRSVneo), from mouse mammary tumor virus(MMTV, i.e. pMSG), from adenovirus (pMT2), from herpes simplex virus(HSV, i.e. pTK2 and pHyg), from bovine papillomavirus (BPV, i.e. pdBPVand pBV-1MTHA), from Epstein-Barr Virus (EBV, i.e. p205 and pHEBo) orany other eukaryotic expression vector known in the art.

[0157] Other methods for providing growth factors to the area oftransplantation include the implantation into the brain in proximity tothe graft of any device which can provide an infusion of the factor tothe surrounding cells.

[0158] In vitro Models of CNS Development, Function and Dysfunction, andMethods for Screening Effects of Drugs on Neural Cells

[0159] Neural stem cell progeny cultured in vitro can be used for thescreening of potential neurologically therapeutic compositions. Thesecompositions can be applied to cells in culture at varying dosages, andthe response of the cells monitored for various time periods. Physicalcharacteristics of the cells can be analyzed by observing cell andneurite growth with microscopy. The induction of expression of new orincreased levels of proteins such as enzymes, receptors and other cellsurface molecules, or of neurotransmitters, amino acids, neuropeptidesand biogenic amines can be analyzed with any technique known in the artwhich can identify the alteration of the level of such molecules. Thesetechniques include immunohistochemistry using antibodies against suchmolecules, or biochemical analysis. Such biochemical analysis includesprotein assays, enzymatic assays, receptor binding assays, enzyme-linkedimmunosorbant assays (ELISA), electrophoretic analysis, analysis withhigh performance liquid chromatography (HPLC), Western blots, andradioimmune assays (RIA). Nucleic acid analysis such as Northern blotscan be used to examine the levels of mRNA coding for these molecules, orfor enzymes which synthesize these molecules.

[0160] Alternatively, cells treated with these pharmaceuticalcompositions can be transplanted into an animal, and their survival,ability to form neuronal connections, and biochemical and immunologicalcharacteristics examined as previously described.

[0161] For the preparation of CNS models, neural stem cells and stemcell progeny are proliferated using the methods described above. Uponremoval of the proliferation-inducing growth factor, proliferation ofmultipotent neural stem cells ceases. The neurospheres can bedifferentiated using the methods described above, for example byadhering the neurospheres to a substrate such as poly-ornithine-treatedplastic or glass where the precursor cells begin to differentiate intoneurons and glial cells. Thus, the proliferation-inducing growth factoracts as an extrinsic signaling molecule that can be added or removed atwill to control the extent of proliferation.

[0162] When the proliferation-inducing growth factor is removed, thegrowth-factor responsive stem cell progeny can be co-cultured on afeeder layer. Many types of feeder layers may be used, such asfibroblasts, neurons, astrocytes, oligodendrocytes, tumor cell lines,genetically altered cell lines or any cells or substrate with bioactiveproperties. The feeder layer generally produces a broader range ofphenotypes. In this instance, the feeder layer acts as a substrate andsource of both membrane bound and soluble factors that induce and alterthe differentiation of the stem cell-generated progeny. Compared to amore inert substance, such as poly-L-ornithine, an astrocyte feederlayer, for example, induces a broader range of neuronal phenotypes asdetermined by indirect immunocytochemistry at 7 DIV. When differentiatedon a poly-L-ornithine coated substrate with 1% FBS, neuronal phenotypesare almost exclusively GABAergic or substance P-ergic. Whendifferentiated on an astrocyte feeder layer, in addition to GABAergicand substance P-ergic neurons, somatostatin, neuropeptide Y (NPY),glutamate and met-enkephalin-containing neurons are present. Theastrocytes can be derived from tissue obtained from various brainregions such as the striatum, cortex and spinal cord.

[0163] Once the growth factor is removed, the culture medium may containserum such as 0.5-1.0% FBS. Serum tends to support the differentiationprocess and enhance cell survival, especially when the differentiatingcells are grown at a low density. However, it is possible to culture anddifferentiate the cells using defined conditions.

[0164] Within 1-3 days after removal of the growth factor and placing ofthe cell in conditions that support differentiation and survival, mostor all of the precursor cells begin to lose immunoreactivity for nestinand begin to express antigens specific for neurons, astrocytes oroligodendrocytes. The identification of neurons is confirmed usingimmunoreactivity for the neuron-specific markers previously mentioned.

[0165] The precursor cells described above can be used in methods ofdetermining the effect of a biological agents on neural cells The term“biological agent” refers to any agent, such as a virus, protein,peptide, amino acid, lipid, carbohydrate, nucleic acid, nucleotide,drug, pro-drug or other substance that may have an effect on neuralcells whether such effect is harmful, beneficial, or otherwise.Biological agents that are beneficial to neural cells are referred toherein as “neurological agents”, a term which encompasses anybiologically or pharmaceutically active substance that may provepotentially useful for the proliferation, differentiation or functioningof CNS cells or treatment of neurological disease or disorder. Forexample, the term may encompass certain neurotransmitters,neurotransmitter receptors, growth factors, growth factor receptors, andthe like, as well as enzymes used in the synthesis of these agents.

[0166] Examples of biological agents include growth factors such asFGF-1, FGF-2, EGF and EGF-like ligands, TGFα, IGF-1, NGF, PDGF, andTGFβs; trophic factors such as BDNF, CNTF, and glial-derivedneurotrophic factor (GDNF); regulators of intracellular pathwaysassociated with growth factor activity such as phorbol 12-myristate13-acetate, staurosporine, CGP-41251, tyrphostin, and the like; hormonessuch as activin and TRH; various proteins and polypeptides such asinterleukins, the Bcl-2 gene product, bone morphogenic protein (BMP-2),macrophage inflammatory proteins (MIP-1α, MIP-1β and MIP-2);oligonucleotides such as antisense strands directed, for example,against transcripts for EGF receptors, FGF receptors, and the like;heparin-like molecules such as heparan sulfate; and a variety of othermolecules that have an effect on neural stem cells or stem cell progenyincluding amphiregulin, retinoic acid, and tumor necrosis factor alpha(TNFα).

[0167] To determine the effect of a potential biological agent on neuralcells, a culture of precursor cells derived from multipotent stem cellscan be obtained from normal neural tissue or, alternatively, from a hostafflicted with a CNS disease or disorder such as Alzheimer's Disease,Parkinson's Disease, or Down's Syndrome. The choice of culture willdepend upon the particular agent being tested and the effects one wishesto achieve Once the cells are obtained from the desired donor tissue,they are proliferated in vitro in the presence of aproliferation-inducing growth factor.

[0168] The ability of various biological agents to increase, decrease ormodify in some other way the number and nature of the stem cell progenyproliferated in the presence of EGF or other proliferative factor can bescreened on cells proliferated by the methods described in Examples 1-6.For example, it is possible to screen for biological agents thatincrease the proliferative ability of progenitor cells which would beuseful for generating large numbers of cells for transplantationpurposes. It is also possible to screen for biological agents whichinhibit precursor cell proliferation. In these studies precursor cellsare plated in the presence of the biological factor(s) of interest andassayed for the degree of proliferation which occurs. The effects of abiological agent or combination of biological agents on thedifferentiation and survival of progenitor cells and their progeny canbe determined. It is possible to screen neural cells which have alreadybeen induced to differentiate prior to the screening. It is alsopossible to determine the effects of the biological agents on thedifferentiation process by applying them to precursor cells prior todifferentiation. Generally, the biological agent will be solubilized andadded to the culture medium at varying concentrations to determine theeffect of the agent at each dose. The culture medium may be replenishedwith the biological agent every couple of days in amounts so as to keepthe concentration of the agent somewhat constant.

[0169] Changes in proliferation are observed by an increase or decreasein the number of neurospheres that form and/or an increase or decreasein the size of the neurospheres (which is a reflection of the rate ofproliferation—determined by the numbers of precursor cells perneurosphere). Thus, the term “regulatory factor” is used herein to referto a biological factor that has a regulatory effect on the proliferationof stem cells and/or precursor cells. For example, a biological factorwould be considered a “regulatory factor” if it increases or decreasesthe number of stem cells that proliferate in vitro in response to aproliferation-inducing growth factor (such as EGF). Alternatively, thenumber of stem cells that respond to proliferation-inducing factors mayremain the same, but addition of the regulatory factor affects the rateat which the stem cell and stem cell progeny proliferate. Aproliferative factor may act as a regulatory factor when used incombination with another proliferative factor For example, theneurospheres that form in the presence of a combination of bFGF and EGFare significantly larger than the neurospheres that form in the presenceof bFGF alone, indicating that the rate of proliferation of stem cellsand stem cell progeny is higher.

[0170] Other examples of regulatory factors include heparan sulfate,TGFBs, activin, BMP-2, CNTF, retinoic acid, TNFα, MIP-1α, MIP-1β, MIP-2,NGF, PDGF, interleukins, and the Bcl-2 gene product. Antisense moleculesthat bind to transcripts of proliferative factors and the transcriptsfor their receptors also regulate stem cell proliferation. Other factorshaving a regulatory effect on stem cell proliferation include those thatinterfere with the activation of the c-fos pathway (an intermediateearly gene, known to be activated by EGF), including phorbol 12myristate 13-acetate (PMA; Sigma), which up-regulates the c-fos pathwayand staurosporine (Research Biochemical International) and CGP-41251(Ciba-Geigy), which down regulate c-fos expression and factors, such astyrphostin [Fallon, D et al., Mol. Cell Biol., 11(5): 2697-2703 (1991)]and the like, which suppress tyrosine kinase activation induced by thebinding of EGF to its receptor.

[0171] Preferred regulatory factors for increasing the rate at whichneural stem cell progeny proliferate in response to FGF are heparansulfate and EGF. Preferred regulatory factors for decreasing the numberof stem cells that respond to proliferative factors are members of theTGFβ family, interleukins, MIPs, PDGF, TNFα, retinoic acid (10⁻⁶ M) andCNTF. Preferred factors for decreasing the size of neurospheresgenerated by the proliferative factors are members of the TGFβ family,retinoic acid (10⁻⁶ M) and CNTF.

[0172] The regulatory factors are added to the culture medium at aconcentration in the range of about 10 pg/ml to 500 ng/ml, preferablyabout 1 ng/ml to 100 ng/ml. The most preferred concentration forregulatory factors is about 10 ng/ml. The regulatory factor retinoicacid is prepared from a 1 mM stock solution and used at a finalconcentration between about 0.01 μM and 100 μM, preferably between about0.05 to 5 μM. Preferred for reducing the proliferative effects of EGF orbFGF on neurosphere generation is a concentration of about 1 μM ofretinoic acid. Antisense strands, can be used at concentrations fromabout 1 to 25 μM. Preferred is a range of about 2 to about 7 μM. PMA andrelated molecules, used to increase proliferation, may be used at aconcentration of about 1 μg/ml to 500 μg/ml, preferably at aconcentration of about 10 μg/ml to 200 μg/ml. The glycosaminoglycan,heparan sulfate, is a ubiquitous component on the surface of mammaliancells known to affect a variety of cellular processes, and which bindsto growth factor molecules such as FGF and amphiregulin, therebypromoting the binding of these molecules to their receptors on thesurfaces of cells. It can be added to the culture medium in combinationwith other biological factors, at a concentration of about 1 ng/ml to 1mg/ml; more preferred is a concentration of about 0.2 μg/ml to 20 μg/ml,most preferred is a concentration of about 2 μg/ml.

[0173] Using these screening methods, it is possible to screen forpotential drug side-effects on pre- and post-natal CNS cells by testingfor the effects of the biological agents on stem cell and progenitorcell proliferation and on progenitor cell differentiation or thesurvival and function of differentiated CNS cells. The proliferatedprecursor cells are typically plated at a density of about 5-10×10⁶cells/ml. If it is desired to test the effect of the biological agent ona particular differentiated cell type or a given make-up of cells, theratio of neurons to glial cells obtained after differentiation can bemanipulated by separating the different types of cells. For example, theO4 antibody (available from Boerhinger Mannheim) binds tooligodendrocytes and their precursors. Using a panning procedure,oligodendrocytes are separated out. Astrocytes can be panned out after abinding procedure using the RAN 2 antibody (available from ATCC).Tetanus toxin (available from Boerhinger Mannheim) can be used to selectout neurons. By varying the trophic factors added to the culture mediumused during differentiation it is possible to intentionally alter thephenotype ratios. Such trophic factors include EGF, FGF, BDNF, CNTF,TGFα, GDNF, and the like. For example, FGF increases the ratio ofneurons, and CNTF increases the ratio of oligodendrocytes. Growing thecultures on beds of glial cells obtained from different CNS regions willalso affect the course of differentiation as described above. Thedifferentiated cultures remain viable (with phenotype intact) for atleast a month

[0174] The effects of the biological agents are identified on the basisof significant difference relative to control cultures with respect tocriteria such as the ratios of expressed phenotypes (neurons: glialcells, or neurotransmitters or other markers), cell viability andalterations in gene expression. Physical characteristics of the cellscan be analyzed by observing cell and neurite morphology and growth withmicroscopy. The induction of expression of new or increased levels ofproteins such as enzymes, receptors and other cell surface molecules, orof neurotransmitters, amino acids, neuropeptides and biogenic amines canbe analyzed with any technique known in the art which can identify thealteration of the level of such molecules. These techniques includeimmunohistochemistry using antibodies against such molecules, orbiochemical analysis. Such biochemical analysis includes protein assays,enzymatic assays, receptor binding assays, enzyme-linked immunosorbantassays (ELISA), electrophoretic analysis, analysis with high performanceliquid chromatography (HPLC), Western blots, and radioimmune assays(RIA). Nucleic acid analysis such as Northern blots and PCR can be usedto examine the levels of mRNA coding for these molecules, or for enzymeswhich synthesize these molecules.

[0175] The factors involved in the proliferation of stem cells and theproliferation, differentiation and survival of stem cell progeny, and/ortheir responses to biological agents can be isolated by constructingcDNA libraries from stem cells or stem cell progeny at different stagesof their development using techniques known in the art. The librariesfrom cells at one developmental stage are compared with those of cellsat different stages of development to determine the sequence of geneexpression during development and to reveal the effects of variousbiological agents or to reveal new biological agents that alter geneexpression in CNS cells. When the libraries are prepared fromdysfunctional tissue, genetic factors may be identified that play a rolein the cause of dysfunction by comparing the libraries from thedysfunctional tissue with those from normal tissue. This information canbe used in the design of therapies to treat the disorders. Additionally,probes can be identified for use in the diagnosis of various geneticdisorders or for use in identifying neural cells at a particular stagein development.

[0176] Electrophysiological analysis can be used to determine theeffects of biological agents on neuronal characteristics such as restingmembrane potential, evoked potentials, direction and ionic nature ofcurrent flow and the dynamics of ion channels. These measurements can bemade using any technique known in the art, including extracellularsingle unit voltage recording, intracellular voltage recording, voltageclamping and patch clamping. Voltage sensitive dyes and ion sensitiveelectrodes may also be used.

[0177] The following examples are presented in order to more fullyillustrate the preferred embodiments of the invention. They should in noway be construed, however, as limiting the scope of the invention, asdefined by the appended claims.

EXAMPLE 1 Dissociation of Embryonic Neural Tissue

[0178] 14-day-old CD₇ albino mouse embryos (Charles River) weredecapitated and the brain and striata were removed using sterileprocedure. Tissue was mechanically dissociated with a fire-polishedPasteur pipette into serum-free medium composed of a 1:1 mixture ofDulbecco's modified Eagle's medium (DMEM) and F-12 nutrient (Gibco).Dissociated cells were centrifuged at 800 r.p.m. for 5 minutes, thesupernatant aspirated, and the cells resuspended in DMEM/F-12 medium forcounting.

EXAMPLE 2 Dissociation of Adult Neural Tissue

[0179] Brain tissue from juvenile and adult mouse brain tissue wasremoved and dissected into 500 μm sections and immediately transferredinto low calcium oxygenated artificial cerebrospinal fluid (low Ca²⁺aCSF) containing 1.33 mg/ml trypsin, 0.67 mg/ml hyaluronidase, and 0.2mg/ml kynurenic acid. Tissue was stirred in this solution for 90 minutesat 32° C.-35° C. aCSF was poured off and replaced with fresh oxygenatedaCSF for 5 minutes. Tissue was transferred to DMEM/F-12/10% hormonesolution containing 0.7 mg/ml ovomucoid and triturated with a firepolished pasteur pipette. Cells were centrifuged at 400 rpm. for 5minutes, the supernatant aspirated and the pelleted cells resuspended inDMEM/F-12/10% hormone mix

EXAMPLE 3 Proliferation of Neural Stem Cells on Substrates

[0180] 2500 cells/cm² prepared as in Example 1 were plated onpoly-L-ornithine-coated (15 μg/ml;Siqma) glass coverslips in 24 wellNunclon (0.5 ml/well) culture dishes The culture medium was a serum-freemedium composed of DMEM/F-12 (1:1) including glucose (0.6%), glutamine(2 EM), sodium bicarbonate (3 mM), and HEPES(4-[2hydroxyethyl]-1-piperazineethanesulfonic acid) buffer (5 mM) (allfrom Sigma except glutamine [Gibco]). A defined hormone mix and saltmixture (Sigma) that included insulin (25 μg/ml), transferrin (100μg/ml), progesterone (20 nM), putrescine (60 EM), and selenium chloride(30 nM) was used in place of serum. Cultures contained the above medium,hereinafter referred to as “Complete Medium” together with 16-20 ng/mlEGF (purified from mouse sub-maxillary, Collaborative Research) or TGFα(human recombinant, Gibco). After 10-14 days in vitro, media (DMEM onlyplus hormone mixture) and growth factors were replaced. This mediumchange was repeated every two to four days. The number of survivingcells at 5 days in vitro was determined by incubating the coverslips in0.4% trypan blue (Gibco) for two minutes, washing with phosphatebuffered saline (PBS, pH 7.3) and counting the number of cells thatexcluded dye with a Nikon Diaphot inverted microscope.

EXAMPLE 4 Proliferation of Embryonic Mouse Neural Stem Cells inSuspension

[0181] Dissociated mouse brain cells prepared as in Examples 1 and 2 (at1×10⁵ cell/ml) were suspended in Complete Medium with 20 ng/ml of EGF orTGFα. Cells were seeded in a T25 culture flask and housed in anincubator at 37° C., 100% humidity, 95% air/5% CO₂. Cells began toproliferate within 3-4 days and due to a lack of substrate lifted offthe floor of the flask and continued to proliferate in suspensionforming clusters of undifferentiated cells, referred to herein as“neurospheres”. After 6-7 days in vitro the proliferating clusters(neurospheres) were fed every 2-4 days by gentle centrifugation andresuspension in DMEM with the additives described above.

EXAMPLE 5 Proliferation of Adult Mouse Neural Stem Cells in Suspension

[0182] The striata, including the subependymal region, of female,pathogen-free CD1 albino mice [3 to 18 month old; Charles River (CF1 andCF2 strains yielded identical results)] were dissected and hand cut withscissors into 1-mm coronal sections and transferred into aCSF (pH 7.35,approx. 180 mOsmol), aerated with 95% O₂-5% CO₂ at room temperature.After 15 minutes the tissue sections were transferred to a spinner flask(Bellco Glass) with a magnetic stirrer filled with low-Ca²⁺ aCSF (pH7.35, approx. 180 mOsmol), aerated with 95% O₂-5% CO₂ at 32 to 35° C.,containing 1.33 mg/ml of trypsin (9000 BAEE units/mg), 0.67 mg/ml ofhyaluronidase (2000 units/mg) and 0.2 mg/ml of kynurenic acid. After 90minutes, tissue sections were transferred to normal aCSF for 5 minutesprior to trituration. Tissue was transferred to DMEM/F-12 (A: 1, Gibco)medium containing 0.7 mg/ml ovomucoid (Sigma) and trituratedmechanically with a fire-narrowed pasteur pipet. Cells were plated (1000viable cells per plate) in noncoated 35 mm culture dishes (Costar)containing Complete Medium and EGF [20 ng/ml, purified from mousesub-maxillary gland (Collaborative Research)] or human recombinant(Gibco/BRL). Cells were allowed to settle for 3-10 minutes after whichthe medium was aspirated away and fresh DMEM/F-12/hormone mix/EGF wasadded. After 5-10 days in vitro the number of spheres (neurospheres)were counted in each 35 mm dish.

EXAMPLE 6 Passaging Proliferated Stem Cells

[0183] After 6-7 days in vitro, individual cells in the neurospheresfrom Example 4 were separated by triturating the neurospheres with afire polished pasteur pipette. Single cells from the dissociatedneurospheres were suspended in tissue culture flasks in DMEM/F-12/10%hormone mix together with 20 ng/ml of EGF. A percentage of dissociatedcells began to proliferate and formed new neurospheres largely composedof undifferentiated cells. The flasks were shaken well and neurosphereswere allowed to settle in the bottom corner of the flask. Theneurospheres were then transferred to 50 ml centrifuge tubes andcentrifuged at 300 rpm for 5 minutes. The medium was aspirated off, andthe neurospheres were resuspended in 1 ml of medium containing EGF. Thecells were dissociated with a fire-narrowed pasteur pipette andtriturated forty times. 20 microliters of cells were removed forcounting and added to 20 microliters of Trypan Blue diluted 1:2. Thecells were counted and replated at 50,000 cells/ml. This procedure canbe repeated weekly and results in a logarithmic increase in the numberof viable cells at each passage. The procedure is continued until thedesired number of stem cell progeny is obtained.

EXAMPLE 7 Differentiation of Neural Stem Cell Progeny andImmunocytochemistry

[0184] Cells proliferated from Examples 4 and 6 were induced todifferentiate by maintaining the cells in the culture flasks in thepresence of EGF or TGFα at 20 ng/ml without reinitiating proliferationby dissociation of the neurospheres or by plating on poly-ornithine inthe continued presence of EGF or TGFα.

[0185] Indirect immunocytochemistry was carried out with cells preparedas in Example 3 which had been cultured for 14-30 days in vitro on glasscoverslips. For anti-NSE (or anti-nestin) and anti-GFAPimmunocytochemistry, cells were fixed with 4% paraformaldehyde in PBSand 95% ethanol/5% acetic acid, respectively. Following a 30 minutefixation period, coverslips were washed three times (10 minutes each) inPBS (pH=7.3) and then incubated in the primary antiserum (NSE 1:300,nestin 1:1500 or GFAP 1:100) in PBS/10% normal goat serum/0.3%Triton-X-100) for two hours at 37 C. Coverslips were washed three times(10 minutes each) in PBS and incubated with secondary antibodies(goat-anti-rabbit-rhodamine for anti-NSE or anti-nestin andgoat-anti-mouse-fluorescein for antiGFAP, both at 1:50) for 30 minutesat 37° C. Coverslips were then washed three times (10 minutes each) inPBS, rinsed with water, placed on glass slides and coverslipped usingFluorsave, a mounting medium preferable for use withfluorescein-conjugated antibodies. Fluorescence was detected andphotographed with a Nikon Optiphot photomicroscope.

[0186] Neural stem cell progeny were also differentiated using thefollowing differentiation paradigms. The neurospheres used for eachparadigm were generated as outlined in Examples 4 and 6. All theneurospheres used were passaged at least once prior to theirdifferentiation.

[0187] Paradigm 1—Rapid Differentiation of Neurospheres

[0188] Six to 8 days after the first passage, the neurospheres wereremoved and centrifuged at 400 r.p.m. The EGF-containing supernatant wasremoved and the pellet suspended in EGF-free complete medium containing1% FBS. Neurospheres (approximately 0.5-1.0×10⁶ cells/well) were platedon poly-L-ornithine-coated (15 μg/ml) glass coverslips in 24 well Nuclon(1.0 ml/well) culture dishes. After 24 hours in culture, the coverslipswere transferred to 12 well (Costar) culture dishes containing completemedium containing 0.5% FBS. The medium was changed every 4-7 days. Thisdifferentiation procedure is referred to as the “Rapid DifferentiationParadigm” or RDP.

[0189] Paradigm 2—Differentiation of Dissociated Neurospheres

[0190] Six to 8 days after the first passage, the neurospheres wereremoved and centrifuged at 400 r.p.m. The EGF-containing media wasremoved and the pellet was suspended in EGF-free complete mediumcontaining 1% FBS. The neurospheres were mechanically dissociated intosingle cells with a fire-polished Pasteur pipette and centrifuged at 800r.p.m. for 5 minutes. Between 0.5×10⁶ and 1.0×10⁶ cells were plated onpoly-L-ornithine-coated (15 μg/ml) glass coverslips in 24 well Nuclon(1.0 ml/well) culture dishes. The EGF-free culture medium containing 1%FBS was changed every 4-7 days.

[0191] Paradigm 3—Differentiation of Single Neurospheres

[0192] Neurospheres were washed free of EGF by serial transfers throughchanges of EGF-free medium. A single neurosphere was plated ontopoly-L-ornithine-coated (15 μg/ml) glass coverslips in a 24-well plate.The culture medium used was complete medium with or without 1% FBS. Themedium was changed every 4-7 days.

[0193] Paradigm 4—Differentiation of Single Dissociated Neurospheres

[0194] Neurospheres were washed free of EGF by serial transfers throughchanges of EGF-free medium. A single neurosphere was mechanicallydissociated in a 0.5 ml Eppendorf centrifuge tube and all the cells wereplated onto a 35 mm culture dish. Complete medium was used with orwithout 1% FBS.

[0195] Paradigm 5—Differentiation of Neurospheres Co-Cultured withStriatal Astrocytes

[0196] Neurospheres, derived from striatal cells as described in Example1 were labeled with 5-bromodeoxyuridine (BrdU) and washed free of EGF.An astrocyte feeder layer was generated from striatal tissue ofpostnatal mice (0-24 hours), and plated on poly-L-ornithine-coated glasscoverslips in a 24-well culture dish. When the astrocytes wereconfluent, a dissociated or intact neurosphere was placed on eachastrocyte bed. Complete medium was changed after the first 24 hours andthen every forty-eight hours. When differentiated on an astrocyte feederlayer, in addition to GABAergic and substance P-ergic neurons,somatostatin, NPY, glutamate and methenkephalin-containing neurons werepresent.

EXAMPLE 8 Effect of Growth Factors on Neurosphere Differentiation

[0197] The effects of CNTF, FGF-2, BDNF, and Retinoic Acid onneurosphere differentiation were tested using the differentiationparadigms set forth in Example 7.

[0198] CNTF

[0199] The effect of CNTF was assayed in paradigms 1 and 3. For bothparadigms, CNTF was added either at the beginning of the experiment at aconcentration of 10 ng/ml or daily at a concentration of 1 ng/ml. Inparadigm 1, the addition of CNTF increased the number ofNSE-immunoreactive cells in addition to the number oftau-1-immunoreactive cells, suggesting that CNTF has an effect on theproliferation, survival, or differentiation of neurons. Preliminarytesting with antibodies recognizing the neurotransmitters GABA andsubstance P suggest that there is no increase in the number of cellscontaining these proteins. This suggests that a different neuronalphenotype is being produced.

[0200] Three different antibodies directed against O4,galactocerebroside (GalC) and MBP were used to study the effect of CNTFon the oligodendrocytes of paradigm 1. CNTF had no effect on the numberof O4(+) cells, but there was an increase in the number of GalC(+) andMBP(+) cells compared with the control. Thus it appears that CNTF playsa role in the maturation of oligodendrocytes.

[0201] In one experiment, the neurospheres were differentiated asoutlined in paradigm 1 except that serum was never added to the culturemedium. While the effect of CNTF on neurons and oligodendrocytes was notas apparent as in the presence of serum, there was an increase in theproliferation of flat, protoplasmic astrocytes. Hence, CNTF will affectastrocyte differentiation in various culture conditions.

[0202] In paradigm 3, the addition of CNTF resulted in an increase inthe number of NSE(+) cells.

[0203] BDNF

[0204] The effect of BDNF was tested using Paradigm 3. There was anincrease in the number of NSE(+) neurons per neurosphere. Additionally,there was an increase in the neuronal branching and the migration of theneurons away from the sphere.

[0205] FGF-2

[0206] The effect of FGF-2 was tested using paradigms 2 and 4. Inparadigm 2, 20 ng/ml of FGF-2 was added at the beginning of theexperiment and cells were stained 7 days later. FGF-2 increased thenumber of GFAP(+) cells and the number of NSE(+) cells. This suggeststhat FGF-2 has a proliferative or survival effect on the neurons andastrocytes.

[0207] In paradigm 4, 20 ng/ml of FGF-2 was added at the beginning ofthe experiment and assayed 7-10 days later. FGF-2 induced theproliferation of neural stem cell progeny generated by theEGF-responsive stem cell. It induced two different cell types to divide,neuroblasts and bipotential progenitor cells. The neuroblast produced,on average, 6 neurons while the bipotential cell produced approximately6 neurons and a number of astrocytes.

[0208] In previous studies, it was found that when plated at low density(2500 cells/cm²), addition of EGF up to 7 days in vitro (DIV) couldinitiate proliferation of the stem cell, but not if applied after 7 DIV.Striatal cells (E14, 2500 cell/cm²) were plated in the absence orpresence of 20 ng/ml of FGF-2. After 11 DIV, cultures were washed andmedium containing 20 ng/ml of EGF was added After 4-5 DIV, in culturesthat were primed with FGF-2, greater than 70% of the wells examinedcontained clusters of proliferating cells that developed into colonieswith the morphologic and antigenic properties of the EGF-generatedcells. Cultures that had not been primed with FGF-2 showed noEGF-responsive proliferation. These findings suggest that theEGF-responsive stem cells possess FGF-2 receptors that regulate its longterm survival.

[0209] Retinoic Acid

[0210] The effect of retinoic acid at 10⁻⁷M was tested using paradigm 1.There was an increase in the number of NSE(+) and tau-1(+) cells,suggesting that retinoic acid increases the number of neurons.

EXAMPLE 9 Proliferation of Embryonic Human Neural Stem Cells andDifferentiation of the Neural Stem Cell Progeny

[0211] With approval of the Research Ethical Committee at the Universityof Lund and the Ethics Committee at the University of Calgary, nine 8-12week old human fetuses were obtained by suction abortions. Tissue wasdissected and any identifiable brain regions were removed. Within 4-5days post-dissection, tissue pieces were mechanically dissociated intosingle cells using the procedure of Example 1 and the number of viablecells was counted. About 0.1×10⁶-0.5×10⁶ cells were plated in 35 cm²tissue culture flasks (without substrate pre-treatment) in CompleteMedium with 20 ng/ml of human recombinant EGF (Gibco/BRL).

[0212] Two to three days after plating the cells, the majority of theviable cells had extended processes and had taken on a neuronalmorphology. By seven days in vitro (DIV), the neuronal-like cells beganto die and by 14 DIV nearly all of these cells were dead or dying(determined by the absence of processes, irregular membranes andgranular cytoplasm). A few of the cells (1%) did not extend anyprocesses or flatten nor did they take on an astrocytic morphology,instead these cells remained rounded and by 5 to 7 DIV began to divide.By 10 to 14 DIV, small clusters of cells, attached to the substrate,were identified. During the next 7 to 10 days (17 to 24 DIV), thesesmall clusters continued to grow in size and many remained attached tothe substrate. By 28 to 30 DIV, nearly all the proliferating clustershad lifted off the substrate and were floating in suspension. Whilefloating in suspension, the clusters continued to grow in size and werepassaged after they had been in culture for 30 to 40 days using theprocedure described in Example 6. EGF-responsive cells began toproliferate after a few DIV and formed floating spheres that werepassaged a second time after 30 to 40 DIV.

[0213] Thirty to 60 days after passage two or three, 2-3 ml aliquotscontaining media and pass 2 spheres were taken from the tissue cultureflasks and plated onto 35 mm culture dish. Single spheres were placedonto poly-L-ornithine coated glass coverslips in DMEM/F-12/HM mediumcontaining EGF. Spheres immediately attached to the substrate and withinthe first 24-48 hours cells begin to migrate from the sphere. At 14 DIVcells continued to proliferate and migrate resulting in an increase inthe diameter of the transferred sphere. By 30 DIV, a large number ofcells had been generated from the original sphere and had migrated at asimilar rate from the center producing a concentric circle of associatedcells. At the periphery, the majority of the cells were one cell layerthick while closer to the center there were denser regions of cells.

[0214] Forebrain regions from eight week old tissue produced no spheres,while spheres were observed from hindbrain tissue in two of the foureight week old samples. For the nine week old fetuses, spheres weregenerated from forebrain region in two of the four samples and in two ofthe three hindbrain regions which were received. The twelve week oldfetus contained only hindbrain tissue and spheres were produced.

[0215] Spheres generated from primary culture or pass 1 spheres wereremoved from the tissue culture flask, without inducing differentiation,and plated onto poly-L-ornithine coated glass coverslips in DMEM/F-12/HMmedium for two hours to allow the spheres to attach to the substrate.Coverslips were removed and processed for indirect immunohistochemistry.Immunostaining with antibodies directed against neurofilaments (168 kDa)or GFAP did not identify any immunoreactive (IR) cells. However, nearlyall of the cells were immunoreactive with an antibody that recognizeshuman nestin.

[0216] Thirty to 45 days after being plated onto the poly-L-ornithinecoated substrate, cells were fixed and processed for indirectimmunocytochemical analysis with antibodies directed against: MAP-2,Tau-1, neurofilament 168 kDA, GABA, substance P (neuronal markers); GFAP(astrocyte marker); 04 and MBP (oligodendrocyte markers). Numerous MAP-2and Tau-1-IR cell bodies and processes were identified in addition to alarge number of Tau-1-IR fibers. While there was no indication ofsubstance P immunoreactivity, GABA-IR cell bodies with long branchedprocesses were seen. Neurofilament-IR cells were strongly IR for GFAP.O4-IR cells with an O-2A morphology and an oligodendrocyte morphologywere present. MBP-IR (found on oligodendrocytes) was also seenthroughout the cultures

EXAMPLE 10 Proliferation of Adult Monkey (Rhesus) Neural Stem Cells andDifferentiation of the Neural Stem Cell Progeny

[0217] The conus medularis was removed from an adult male monkey(Rhesus) and hand cut with scissors into 1-mm sections and transferredinto artificial cerebrospinal fluid (aCSF) containing 124 mM NaCl, 5 mMKCl, 1.3 mM MgCl₂, 2 mM CaCl₂, 26 mM NaHCO₃, and 10 mM D-glucose (pH7.35, approx. 280 mOsmol), aerated with 95% O₂-5% CO₂ at roomtemperature. After 15 min, the tissue sections were transferred to aspinner flask (Bellco Glass) with a magnetic stirrer filled withlow-Ca²⁺ aCSF containing 124 mM NaCl, 5 mM KCl, 3.2 mM MgCl₂, 0.1 mMCaCl₂, 26 mM NaHCO₃, and 10 mM D-glucose (pH 7.35, approx. 280 mOsmol),aerated with 95% O₂-5% CO₂ at 32 to 35° C., containing 1.33 mg/ml oftrypsin (9000 BAEE units/mg), 0.67 mg/ml of hyaluronidase (2000units/mg) and 0.2 mg/ml of kynurenic acid. After 90 min, tissue sectionswere transferred to normal aCSF for S min prior to trituration. Tissuewas transferred to DMEM/F-12 (1:1, Gibco) medium containing 0.7 mg/mlovomucoid (Sigma) and triturated mechanically with a fire-narrowedpasteur pipet.

[0218] Cells were plated (1000 viable cells per plate) in non-coated 35mm culture dishes (Costar) containing Complete Medium and 20 ng/ml EGF(human recombinant from Gibco/BRL). After 7 to 10 days in culture,floating spheres were transferred with wide-bore pipets onto laminin (15μg/ml)(Sigma)-coated glass coverslips in 24-well culture dishes. EGF @20ng/ml was added to the medium. Spheres attached to the substrate andcells within the sphere continued to proliferate. After 14 to 21 days invitro (DIV), the cells were probed by indirect immunocytochemistry forthe presence of neuron, astrocytes and oligodendrocytes. All three celltypes were identified.

EXAMPLE 11 Proliferation of Adult Human Neural Stem Cells andDifferentiation of the Neural Stem Cell Progeny

[0219] During a routine biopsy, normal tissue was obtained from a 65year old female patient. The biopsy site was the right frontal lobe, 6mm from the tip of the frontal/anterior horn of the lateral ventricle.The tissue was dissociated using the procedure outlined in Example 2 andcultured in Complete Medium with EGF and FGF-2 (20 ng/ml of each growthhormone), in T25 flasks (Nunclon). The flasks were examined every 2-3days for neurosphere formation. Clonally-derived cells were passagedusing single sphere dissociation: single neurospheres were triturated100 × in sterile aliquot tubes containing 200 μl of themedia/hormone/EGF-FGF-2 solution before culturing in 24- or 96-wellplates. First-passage neurospheres were plated on poly-ornithine andlaminin coated coverslips and allowed to plate down for 14 days inmedia/hormone/EGF+FGF-2. Some first passage neurospheres were plated onlaminin (20 μg/ml) and poly-ornithine coated coverslips in media/hormonemix for 19 hours, then processed for nestin staining as outlined inExample 7. Nestin staining indicated that the neurospheres, prior to theinduction of differentiation (as described below) were nestin positive,indicative of the presence of immature undifferentiated cells.

[0220] Pass one human neurospheres were plated on a laminin coatedsubstrate (see above). After 14 days, the cultures received a mediachange to media/hormone mix plus 1% FBS and were allowed todifferentiate for 7 days. Immunocytochemical analysis was then performedto determine different neural phenotypes. The differentiated cells werefixed with 4% paraformaldehyde in PBS for 20 minutes. The coverslipswere washed three times (five minutes each) in PBS. For triple labelimmunocytochemistry, the cells were permeabilized for 5 minutes in 0.3%Triton-X in PBS followed by 2 washes with PBS. A first set of primaryantibodies, MAP-2 (mouse monoclonal, 1:1000, Boerhinger Mannheim) andGFAP (Rabbit polyclonal, 1:300, BTI), used to determine the presence ofneurons and astrocytes respectively, were mixed in 10% normal goat serumin PBS. The cells were incubated at 37° C. for 2 hours and then washed 3times in PBS. A first set of secondary antibodies, goat anti-mouserhodamine (Jackson Immuno Research) and goat anti-rabbit FITC (IgG,1:100 Jackson Immuno Research) were mixed in PBS. The cells wereincubated for 30 minutes at 37° C. and then rinsed three times with PBS.The second primary antibody, O4 (mouse monoclonal IgM, 1:10) foroligodendrocytes, was mixed in 10% normal goat serum in PBS. The cellswere incubated for 2 hours at 37° C. The second set of secondary goatanti-mouse AMCA IgM (1:100 Jackson Immuno Research) was mixed in PBS andcells were incubated for 30 minutes at 37° C. The cells were then rinsedtwice in PBS and then in double distilled water before mounting withFluorosave.

EXAMPLE 12 Screening for the trkB Receptor on Neural Stem Cell Progeny

[0221] The expression of the trk family of neurotrophin receptors inEGF-generated neurospheres was examined by northern blot analysis. TotalmRNA was isolated from mouse and rat striatal EGF-generatedneurospheres. Both rat and mouse neurospheres expressed high levels oftrkB receptor mRNA, but did not express trk nor trkC mRNA. Inpreliminary experiments, single EGF-generated mouse neurospheres wereplated on poly-L-ornithine coated glass coverslips and cultured in theabsence or presence of 10 ng/ml of BDNF. When examined after 14-28 daysin vitro, neurospheres plated in the presence of BDNF contained NSE(+)cells with extensive and highly branched processes; well-developedNSE(+) cells were not observed in the absence of BDNF. Activation of thetrkB receptor on EGF-generated neurospheres may enhance differentiation,survival of and/or neurite outgrowth from newly generated neurons.

EXAMPLE 13 Screening for the GAP-43 Membrane Phosphoprotein on NeuralStem Cell Progeny

[0222] Growth-associated protein (GAP-43) is a nervous system-specificmembrane phosphoprotein which is down-regulated during development.Originally, GAP-43 was though to be neuron-specific, however, recentreports indicate that this protein may be at least transiently expressedduring development in some astrocytes, oligodendrocytes and in Schwanncells. At present, the role of GAP-43 in macroglia is not known. Thetransient expression of GAP-43 in glial cells generated from theEGF-responsive stem cells derived from embryonic and adult murinestriatum was investigated. Glial cell (astrocyte and oligodendrocyte)differentiation was induced by plating neural stem cell progeny in amedium containing 1% FBS with no EGF. The cells were then probed withspecific antibodies for GAP-43, nestin, GFAP, 04, and GalC. In order toidentify cells expressing GAP-43, the antibodies were pooled in variouscombinations using dual-label immunofluorescence methods.

[0223] During the first two days post plating, there was a low tomoderate level of GAP-43 expression in almost all cells (flat, bipolarand stellate), but by 3-4 days post-plating, the level of GAP-43expression became restricted to the bipolar and stellate cells. At 4days the majority of GAP-43-expressing cells co-labelled with theoligodendrocyte markers 04 and GalC although GFAP and GAP43 wasco-expressed in a number of cells. At one week post-plating however,essentially all of the GFAP-expressing astrocytes no longer expressedGAP-43 while the majority of the O4 and GalC-expressing cells continuedto express GAP-43. At 7-10 days, these oligodendrocytes began to expressMBP and lose the expression of GAP-43. The EGF-responsive stem cells mayrepresent a useful model system for the study of the role of GAP-43 inglial and neuronal development.

EXAMPLE 14 Treatment of Neurodegenerative Disease Using Progeny of HumanNeural Stem Cells Proliferated in vitro

[0224] Cells are obtained from ventral mesencephalic tissue from a humanfetus aged 8 weeks following routine suction abortion which is collectedinto a sterile collection apparatus A 2×4×1 mm piece of tissue isdissected and dissociated as in Example 1. Neural stem cells are thenproliferated as in Example 4. Neural stem cell progeny are used forneurotransplantation into a blood-group matched host with aneurodegenerative disease. Surgery is performed using a BRW computedtomographic (CT) stereotaxic guide. The patient is given localanesthesia suppiemencea with intravenously administered midazolam. Thepatient undergoes CT scanning to establish the coordinates of the regionto receive the transplant. The injection cannula consists of a 17-gaugestainless steel outer cannula with a 19-gauge inner stylet. This isinserted into the brain to the correct coordinates, then removed andreplaced with a 19-gauge infusion cannula that has been preloaded with30 μl of tissue suspension. The cells are slowly infused at a rate of 3μl/min as the cannula is withdrawn. Multiple stereotactic needle passesare made throughout the area of interest, approximately 4 mm apart. Thepatient is examined by CT scan postoperatively for hemorrhage or edema.Neurological evaluations are performed at various post-operativeintervals, as well as PET scans to determine metabolic activity of theimplanted cells.

EXAMPLE 15 Remyelination of Myelin Deficient Rats Using Neural Stem CellProgeny Proliferated in vitro

[0225] Embryonic day 15 (E15) Sprague Dawley rats and E14-15 mice wereobtained and the neural tissue was prepared using the methods describedin Example 1. The cells were suspended in Complete Medium with 16-20ng/ml EGF (purified from mouse submaxillary, Collaborative Research) orTGFα (human recombinant, Gibco). The cells were seeded in a T25 cultureflask and housed in an incubator at 37° C., 100% humidity, 95% air/5%CO₂ and proliferated using the suspension culture method of Example 4.Cells proliferated within 34 days and, due to lack of substrate, liftedoff the floor of the flask and continued to proliferate in suspensionforming neurospheres.

[0226] After 6-8 days in vitro (DIV) the neurospheres were removed,centrifuged at 400 r.p.m. for 2-5 minutes, and the pellet mechanicallydissociated into individual cells with a fire-polished glass pasteurpipet. Cells were replated in the growth medium where proliferation ofthe stem cells and formation of new neurospheres was reinitiated.

[0227] Litters of first day postnatal myelin deficient rats wereanesthetized using ice to produce hypothermia. Myelin deficiency is anX-linked trait and thus only one half of the males in any litter areaffected. Therefore, only the males were used for these studies. Onceanesthetized, a small rostral to caudal incision was made at the levelof the lumbar enlargement. The muscle and connective tissue was removedto expose the vertebral laminae. Using a fine rat tooth forceps, onelamina at the lumbar enlargement was removed and a small cut is made inthe dura mater to expose the spinal cord.

[0228] A stereotaxic device holding a glass pipet was used to inject a 1μl aliquot of the cell suspension (approximately 50,000 cells/μl)described above. The suspension is slowly injected into a single site(although more could be done) in the dorsal columns of the spinal cord.As controls, some of the animals were sham-injected with sterile saline.The animals were marked by clipping either toes or ears to distinguishbetween both experimental groups. Following injection of the cellsuspension, the wound was closed using sutures or stainless steel woundclips and the animals were revived by warming on a surgical heating padand then returned to their mother.

[0229] The animals were allowed to survive for two weeks post-injectionand were then deeply anesthetized with nembutal (150 mg/kg) and perfusedthrough the left ventricle. The spinal cords were removed and the tissueexamined by light and electron microscopy. Patches of myelin were foundin the dorsal columns of the recipients of both rat and mouse cells,indicating that neural stem cells isolated from rat and mouse neuraltissue can differentiate into oligodendroglia and are capable ofmyelination in vivo.

[0230] Because the myelin deficient rat spinal cord is almost completelydevoid of myelin, myelin formed at or near the site of injection isderived from the implanted cells. It is possible that the process ofinjection will allow for the entry of Schwann cells (myelinating cellsof the PNS) into the spinal cord. These cells are capable of formingmyelin within the CNS but can be easily distinguished fromoligodendrocytes using either light microscopy or immunocytochemistryfor CNS myelin elements. There is usually a very small amount of CNSmyelin within the myelin deficient rat spinal cord. This myelin can bedistinguished from normal donor myelin based on the mutation within thegene for the major CNS myelin protein, proteolipid protein (PLP). Themyelin deficient rat myelin is not immunoreactive for PLP while thedonor myelin is.

EXAMPLE 16 Remyelination in Human Neuromyelitis Optica

[0231] Neuromyelitis optica is a condition involving demyelination ofprincipally the spinal cord and optic nerve. Onset is usually acute andin 50% of the cases death occurs within months. The severity ofdemyelination as well as lesion sites can be confirmed by magneticresonance imaging (MRI).

[0232] Neural stem cell progeny are prepared from fetal human tissue bythe methods of Example 9 or 14. Cells are stereotactically injected intothe white matter of the spinal cord in the vicinity of plaques asvisualized by MRI. Cells are also injected around the optic nerve asnecessary. Booster injections may be performed as required.

EXAMPLE 17 Remyelination in Human Pelizaeus-Merzbacher Disease

[0233] Pelizaeus-Merzbacher disease is a condition involvingdemyelination of the CNS. The severity of demyelination as well aslesion sites can be confirmed by magnetic resonance imaging (MRI).

[0234] Neural stem cell progeny are prepared from fetal human tissue bythe methods of Examples 9 or 14. Cells are stereotactically injectedinto the white matter of the spinal cord in the vicinity of plaques asvisualized by MRI. Cells are also injected around the optic nerve asnecessary. Booster injections may be performed as required.

EXAMPLE 18 Genetic Modification of Neural Stem Cell Progeny

[0235] Cells proliferated as in Examples 3 or 4 are transfected withexpression vectors containing the genes for the FGF-2 receptor or theNGF receptor. Vector DNA containing the genes are diluted in 0.1× TE (1mM Tris pH 8.0, 0.1 mM EDTA) to a concentration of 40 μg/ml. 22 μl ofthe DNA is added to 250 μl of 2× HBS (280 mM NaCl, 10 mM KCl, 1.5 mMNa₂HPO₄-2H₂O, 12 mM dextrose, 50 mM HEPES) in a disposable, sterile 5 mlplastic tube. 31 μl of 2 M CaCl₂ is added slowly and the mixture isincubated for 30 minutes at room temperature. During this 30 minuteincubation, the cells are centrifuged at 800 g for 5 minutes at 4° C.The cells are resuspended in 20 volumes of ice-cold PBS and divided intoaliquots of 1×10⁷ cells, which are again centrifuged. Each aliquot ofcells is resuspended in 1 ml of the DNA-CaCl₂ suspension, and incubatedfor 20 minutes at room temperature. The cells are then diluted in growthmedium and incubated for 6-24 hours at 37° C. in 5%-7% CO². The cellsare again centrifuged, washed in PBS and returned to 10 ml of growthmedium for 48 hours.

[0236] The transfected neural stem cell progeny are transplanted into ahuman patient using the procedure described in Example 14, or are usedfor drug screening procedures as described in the examples below.

EXAMPLE 19 Genetic Modify cation of Neural Stem Cell Progeny With aRetrovirus Containing the Bacterial B-Galactosidase Gene

[0237] Neural stem cell progeny were propagated as described in Example4. A large pass-1 flask of neurospheres (4-5 days old) was shaken todislodge the spheres from the flask. The flask was spun at 400 r.p.m.for 3-5 minutes. About half of the media was removed without disturbingthe neurospheres. The spheres and the remaining media were removed,placed into a Falcon 12 ml centrifuge tube, and spun at 600 r.p.m. for3-5 minutes. The remaining medium was removed, leaving a few hundredmicroliters.

[0238] A retrovirus which contained the bacterial B-galactosidase genewas packaged and secreted, in a replication-deficient fashion, by theCRE BAG2 cell line produced by C. Cepko. A day after the CRE cellsreached confluence, the cells were washed with PBS and the retroviruswas collected in DMEM/F12/HM/20 ng/ml EGF for four days. Thevirus-containing media was filtered through a 0.45 μm syringe filter.The neurospheres were resuspended in the virus-containing media,transferred to a large flask, and left in an incubator overnight at 37°C. The next day, the contents of the flask were transferred to a 12 mlcentrifuge tube and spun at 800 r.p.m. The cells were resuspended inEGF-containing media/HM, dissociated into single cells, and counted. Thecells were replated in a large flask at 50,000 cells/ml in a total of 20mls.

[0239] Four days later, transformed cells were selected with G418 at aconcentration of 300 μg/ml. Transformed spheres were plated on apoly-ornithine coated glass coverslip in a 24-well plate. After theneurospheres adhered to the plate, the cells were fixed with 0.1%glutaraldehyde for 5 minutes at 4° C. After the cells were fixed, theywere washed twice with PBS for 10 minutes. The cells were then washedwith 0.1% Triton^(R) in PBS for 10-15 minutes at room temperature. A 1mg/ml X-Gal solution was added to each well and incubated overnight at37° C. After incubation overnight, the cells were washed three timeswith PBS for 10 minutes each and observed for any reaction products. Apositive reaction resulted in a blue color, indicating cells containingthe transferred gene.

EXAMPLE 20 Proliferation of Neutral Stem Cells from Transgenic Mice

[0240] Transgenic mice were produced using standard pronuclear injectionof the MBP-lacZ chimeric gene, in which the promoter for MBP directs theexpression of E. coli B-galactosidase (lacZ). Transgenic animals wereidentified by PCR using oligonucleotides specific for lacZ.

[0241] Neurospheres were prepared from E15 transgenic mice and DNAnegative littermates using the procedures set forth in Example 4. Theneurospheres were propagated in the defined culture medium in thepresence of 20 ng/ml EGF and were passaged weekly for 35 weeks. Forpassaging, the neurospheres were harvested, gently centrifuged at 800RPM, and mechanically dissociated by trituration with a fire-polishedPasteur pipet. At various passages, the cells were induced todifferentiate into oligodendrocytes, astrocytes, and neurons by alteringthe culture conditions The free-floating stem cell clusters were gentlycentrifuged, resuspended in the same base defined medium without EGF andwith 1% FBS and plated on poly ornithine-treated glass coverslips topromote cell attachment. The clusters attach firmly to the glass, andthe cells slow or stop dividing and begin to differentiate.

[0242] The identification of various cell types was accomplished usingimmunofluorescence microscopy with antibodies specific for neurons(MAP-2, NF-L, and NF-M), astrocytes (GFAP) and oligodendrocytes andoligodendrocyte precursors (A2B5, O₁, O₄, Gal C, and MBP). One to 14days post-plating, the cells on the coverslips were incubated unfixed,for 30 minutes at room temperature with the primary antibodies O1, O4,GalC, and A2B5 (supernatants) diluted in minimal essential medium with5% normal goat serum and 25 mM HEPES buffer, pH 7.3 (MEM-HEPES, NGS).Following the primary antibodies, the coverslips were gently washed 5times in rhodamine-conjugated secondary antibodies (Sigma) diluted inMEM-HEPES, NGS. The coverslips were then washed 5 times in MEM-HEPES andfixed with acid alcohol (5% glacial acetic acid/95% ethanol) at −20° C.The coverslips were then washed 5 times with MEM-HEPES, and eithermounted and examined using fluorescence microscopy or immmunoreactedwith rabbit polyclonal antisera raised against GFAP, nestin, MBP, orproteolipid protein (PLP). When subjected to a second round ofimmunolabeling, the coverslips were incubated first for 1 hour with 5%normal goat serum (NGS) in 0.1 M phosphate buffer with 0.9% NaCl at pH7.4 (PBS) and then incubated in rabbit primary antibodies diluted in NGSfor 1-2 hours at room temperature. The coverslips were washed 3 timeswith PBS and then incubated with the appropriate secondary antibodyconjugates diluted in NGS, washed again with PBS and then finallymounted on glass microscope slides with Citifluor antifadent mountingmedium and examined using a fluorescence microscope. In cases were theywere not incubated first with the monoclonal antibody supernatants, thecoverslips were fixed for 20 minutes with 4% paraformaldehyde in PBS (pH7.4), washed with PBS, permeabilized with 100% ethanol, washed againwith PBS and incubated with 5% NGS in PBS for 1 hour. The primaryantibodies and secondary antibody conjugates were applied as outlinedabove.

[0243] The neural stem cells derived from the transgenic animals wereindistinguishable from non transgenic stem cells in their potential fordifferentiation into neurons, astrocytes, and oligodendrocytes. The MBPpromoter directed the expression of the B-galactosidase reporter gene ina cell-specific and developmentally appropriate fashion. The transgeneexpression is highly stable as oligodendrocytes derived from latepassage MBP-lacZ neurospheres (20 passages), expressed theB-galactosidase gene. Thus, transgenically marked neurospheres arelikely to be an excellent source of cells for glial celltransplantation.

EXAMPLE 21 Genetic Modification of Neural Stem Cell Progeny UsingCalcium Phosphate Transfection

[0244] Neural stem cell progeny are propagated as described in Example4. The cells are then infected using a calcium phosphate transfectiontechnique. For standard calcium phosphate transfection, the cells aremechanically dissociated into a single cell suspension and plated ontissue culture-treated dishes at 50% confluence (50,000-75,000cells/cm²) and allowed to attach overnight.

[0245] The modified calcium phosphate transfection procedure isperformed as follows: DNA (15-25 μg) in sterile TE buffer (10 mM Tris,0.25 mM EDTA, pH 7.5) diluted to 440 μl with TE, and 60 μl of 2 M CaCl₂(pH to 5.8 with 1 M HEPES buffer) is added to the DNA/TE buffer. A totalof 500 μl of 2× HeBS (HEPES-Buffered saline; 275 mM NaCl, 10 mM KCl, 1.4mM Na₂HPO₄, 12 mM dextrose, 40 mM HEPES buffer powder, pH 6.92) is addeddropwise to this mix. The mixture is allowed to stand at roomtemperature for 20 minutes. The cells are washed briefly with 1× HeBSand 1 ml of the calcium phosphate precipitated DNA solution is added toeach plate, and the cells are incubated at 37° for 20 minutes. Followingthis incubation, 10 mls of complete medium is added to the cells, andthe plates are placed in an incubator (37° C., 9.5% CO₂) for anadditional 3-6 hours. The DNA and the medium are removed by aspirationat the end of the incubation period, and the cells are washed 3 timeswith complete growth medium and then returned to the incubator.

EXAMPLE 22 Genetically Modified Neural Stem Cell Progeny Expressing NGF

[0246] Using either the recombinant retrovirus or direct DNAtransfection technique, a chimeric gene composed of the human CMVpromoter directing the expression of the rat NGF gene is introduced intothe neurosphere cells. In addition, the vector includes the E. colineomycin resistance gene driven off of a viral promoter. After 2 weeksof G418 selection, the cells are cloned using limiting dilution in96-multi-well plates and the resulting clones are assayed forneurotrophin protein expression using a neurotrophin receptor (trkfamily) autophosphorylation bioassay.

[0247] Clones expressing high levels of NGF are expanded in T-flasksprior to differentiation. The cells are then removed from theEGF-containing complete medium and treated with a combination of serumand a cocktail of growth factors to induce astrocyte differentiation.The astrocytes are again assayed for NGF expression to ensure that thedifferentiated cells continue to express the trophic factors. Astrocytesthat secrete NGF are then injected into fimbria/fornix lesioned ratbrains immediately post-lesioning in order to protect the cholinergicneurons. Control astrocytes that do not secrete NGF are injected intosimilarly lesioned animals. The sparing of cholinergic neurons in thelesion model is assessed using immunocytochemistry for ChAT, the markerfor these cholinergic neurons.

EXAMPLE 23 Genetically Modified Neural Stem Cell Progeny Expressing CGAT

[0248] Recently, a novel chromaffin granule amine transporter (CGAT)cDNA has been described by Liu et al. (Cell. 70:539-551 (1992)), whichaffords resistance to the neurotoxin MPP+ in Chinese hamster ovary (CHO)cells in vitro. Because dopaminergic neurons from the substantia nigraare specifically killed by MPP+, CGAT gene expression in geneticallymodified neural stem cell progeny may improve viability of the cellsafter they are implanted into the Parkinsonian brain. Neural stem cellprogeny are propagated as in Example 4. The cells are mechanicallydissociated and plated on plastic dishes and infected with a retroviruscontaining the CGAT cDNA. The expression of the CGAT cDNA (Liu et al.supra) is directed by a constitutive promoter (CMV or SV40, or aretroviral LTR) or a cell-specific promoter (TH or other dopaminergic orcatecholaminergic cell-specific regulatory element or the like). Thecells are screened for the expression of the CGAT protein. Selectedcells can then be differentiated in vitro using a growth factor or acombination of growth factors to produce dopaminergic orpre-dopaminergic neurons.

EXAMPLE 24 3H-Thymidine Kill Studies Identify Presence of ConstitutivelyProliferating Population of Neural Cells in Subependymal Region

[0249] Adult male CD1 mice received a series of intraperitonealinjections of 3H-thymidine (0.8 ml per injection, specific activity45-55 mCi/mmole, ICN Biomedical) on day 0 (3 injections, 1 every 4hours) in order to kill the constitutively proliferating subependymalcells. On day 0.5, 1, 2, 4, 6, 8 or 12, animals received 2 BrdUinjections 1 hour apart (see Example 25) and were sacrificed 0.5 hourafter the last injection.

[0250] It was observed that 10% of the cells were proliferating on day 1post-kill, and by 8 days the number of proliferating cells had reached85%, which was not statistically significantly different from controlvalues. Animals were sacrificed and the brains were removed andprocessed as described in Example 10.

[0251] In a second group of animals, 3H-thymidine injections were givenon day 0 (3 injections, 1 every 4 hours), followed by an identicalseries of injections on day 2 or 4. Animals were allowed to survive for8 days following the second series of injections (days 9, 10 and 12respectively) at which time they received 2 injections of BrdU and weresacrificed 0.5 hours later. Animals were sacrificed and the brains wereremoved and processed as described in Example 25.

[0252] After the second series of injections on day 2 only 45% of theproliferating population had returned relative to control values. Thisindicates that the second series of injections given on day 2 had killedthe stem cells as they were recruited to the proliferating mode. Thesecond series of injections given on day 4 resulted in a return tocontrol values by day 8 suggesting that by this time, the stem cellswere no longer proliferating and hence were not killed by the day 4series of injections.

EXAMPLE 25 BrdU Labeling Studies Identify Presence of ConstitutivelyProliferating Population of Neural Cells in Subependymal Region

[0253] Adult male CD1 mice (25-30 g, Charles River) were injectedintraperitoneally i.p.) with bromodeoxyuridine (BrdU, Sigma, 65 mg/kg)every 2 hours for a total of 5 injections in order to label all of theconstitutively proliferating cells in the subependyma lining the lateralventricles in the forebrain. One month later, animals were sacrificedwith an overdose of sodium pentobarbital and transcardially perfusedusing 4% paraformaldehyde. The brains were removed and post-fixedovernight in 4% paraformaldehyde with 20% sucrose. Brain sections werecut on a cryostat (30 um) and collected in a washing buffer [0.1 Mphosphate buffered saline (PBS) pH 7.2 with 1% normal horse serum and0.3% Triton X-100]. Sections were incubated in 1M HCl at 60° C. for 0.5hours then washed 3 times (10 minutes each) in washing buffer. Followingthe final wash, sections were incubated in anti-BrdU (Becton Dickinson,1:25) for 45 hours at 4° C. After incubation in the primary antibody,sections were washed 3 times and incubated for 1 hours in biotinylatedhorse-anti-mouse secondary antibody (Dimension Lab, 1:50) at roomtemperature followed by another 3 washes. The sections were thenincubated for 1 hour in avidin conjugated FITC (Dimension Lab, 1:50) atroom temperature and washed a final 3 times. Sections were mounted ongelatin coated slides, air-dried and coverslipped with Fluoromount.Slides were examined for BrdU positive cells using a NIKON fluorescentmicroscope. The number of BrdU positive cells was counted within thesubependyma surrounding the lateral ventricles in 8 samples in sectionsbetween the closing of the corpus callosum rostrally and the crossing ofthe anterior commissure caudally. It was found that 31 days followingthe series of BrdU injections, 3% of the subependymal cells were stilllabeled compared to control animals sacrificed immediately following theseries of injections (control 100%).

EXAMPLE 26 3H-Thymidine Kill Studies Identify Presence of RelativelyQuiescent Neural Stem Cells in Sutbependymal Region

[0254] Adult male CD1 mice were divided into 4 groups. Group A animalsreceived a series of 3H-thymidine injections on day 0 (3 injections, 1every 4 hours). Animals in groups B and C received a series of3H-thymidine injections on day 0 followed by a second series ofinjections on day 2 or 4. Group D animals received injections ofphysiological saline instead of 3H-thymidine over the same time courseas group A. Animals from all groups were sacrificed by cervicaldislocation 16-20 hours following the last series of injections. Brainswere removed and neural tissue obtained from the subependyma surroundingthe lateral ventricles in the forebrain was dissociated and the neuralcells cultured as described in Example 5. At 6 and 8 days in vitro, thetotal number of spheres was counted in each of the 35 mm wells.

[0255] Control animals that received a series of saline injectionsformed the same number of spheres as animals that received 3H-thymidineon day 0 (which kills the normally proliferating subependymal cells).This indicates that the constitutively proliferating subependymal cellsare not the source of stem cells isolated in vitro. Animals thatreceived a second series of injections on day 2 formed 45% the number ofspheres (similar to the number of proliferating subependymal cellsobserved in vivo). When a second series of injections was done on day 4,the number of spheres formed in vitro was not significantly differentfrom control values, again correlating with the in vivo findings. Takentogether, this data indicates that the multipotent spheres, which areisolated in vitro in the presence of EGF, are formed from the relativelyquiescent stem cell population within the subependyma in vivo.

EXAMPLE 27 In Vivo Proliferation of Neural Stem Cells of LateralVentricle

[0256] A replication incompetent retrovirus containing theβ-galactosidase gene [as described in Walsh and Cepko, Science 241:1342,(1988)] was injected into the forebrain lateral ventricles of CD1 adultmale mice (25-30 g from Charles River). The injected retrovirus washarvested from the BAG cell line (ATCC CRL-9560) according to the methodof Walsh and Cepko (supra). Mice were anesthetized using 65 mg/kg, i.p.sodium pentobarbital. Unilateral stereotactic injections of 0.2-1.0 μlof retrovirus were injected into the lateral ventricle using a 1 μlHamilton syringe. The coordinates for injection were AP+4.2 mm anteriorto lambda, L±0.7 mm, and DV −2.3 mm below dura, with the mouth bar at −2mm below the interaural line.

[0257] On the same day as, one day, or six days following the retrovirusinjection, an infusion cannulae attached to a 0.5 μl/hour ALZET osmoticmini-pumps filled with 3.3-330 μg/ml of EGF were surgically implantedinto the lateral ventricles at the identical stereotactic coordinates asstated above. The infusion cannula kits were obtained from ALZA. Theinfusion cannulae were cut to 2.7 mm below the pedestal. The pumps weresecured to the mouse skull by use of acrylic cement and a skull screwcontralateral and caudal to the injection site. The osmotic mini-pumpwas situated subcutaneously under and behind the armpit of the leftfront paw and connected to the infusion cannula by the means ofpolyethylene tubing.

[0258] Six days following initiation of EGF infusion the animals weresacrificed with an overdose of sodium pentobarbital. Mice weretranscardially perfused with 2% buffered paraformaldehyde, and thebrains were excised and post fixed overnight with 20% sucrose in 2%buffered paraformaldehyde. Coronal slices were prepared with −20 celsiuscryostat sectioning at 30 μm. Slices were developed for β-galhistochemistry as per Morshead and Van der Kooy (supra).

[0259] Under these conditions, regardless of the day post retrovirusinjection, infusion of EGF resulted in an expansion of the population ofβ-gal labelled cells from an average of 20 cells per brain up to anaverage of 150 cells per brain and the migration of these cells awayfrom the lining of the lateral ventricles. Infusion of FGF-2 at 33 μg/mlresulted in an increase in the number of β-gal labelled cells, but thisincrease was not accompanied by any additional migration. Infusion ofEGF and FGF together resulted in an even greater expansion of thepopulation of β-gal labelled cells from 20 cells per brain to an averageof 350 cells per brain.

[0260] These results indicate that FGF may be a survival factor forrelatively quiescent stem cells in the subependyma layer, whereas EGFmay act as a survival factor for the normally dying progeny of theconstitutively proliferating population. The synergistic increase inβ-galactosidase cell number when EGF and FGF are infused togetherfurther reflects the direct association between the relatively quiescentstem cell and the constitutively proliferating progenitor cell.

EXAMPLE 28 In Vivo Proliferation of Neural Stem Cells of the Third andFourth Ventricles and the Central Canal

[0261] A retroviral construct containing the β-galactosidase gene ismicroinjected (as in Example 27) into the III ventricle of thediencephalon, IV ventricle of the brain stem and central canal of thespinal cord. Minipumps containing EGF and FGF are then used tocontinuously administer growth factors for six days (as in Example 27)into the same portion of the ventricular system that the retroviralconstruct was administered. This produces an increase in the number ofβ-galactosidase producing cells which survive and migrate out into thetissue near the III ventricle, IV ventricle and central canal of thespinal cord forming new neurons and glia.

EXAMPLE 29 In Vivo Modification and Proliferation of Neural Stem Cellsand Differentiation of Neural Stem Cell Progeny of the Lateral Ventricle

[0262] A retroviral construct containing the TH gene as well as theβ-galactosidase gene is microinjected into the adult lateral ventricleas in Example 27. Minipumps containing EGF, FGF, or EGF and FGF togetherare then used to continuously administer the growth factor(s) into thelateral ventricle for 6 days as in Example 27. As the infectedsubependymal cells migrate out into the striatum they differentiate intoneuronal cells that produce dopamine as measured directly byimmunofluorescence with an antibody and (from a direct functional assay)by the ability to overcome the rotational bias produced by unilateral6-hydroxydopamine lesions.

EXAMPLE 30 In Vivo Infusion of Growth Factors into Ventricles to ObtainElevated Numbers of Neural Stem Cells

[0263] Adult male CD₁ albino mice (30-35 g) from Charles River wereanaesthetized with sodium pentobarbital (0.40 mL of a 10% solution) andplaced in a stereotaxic apparatus. The dorsal aspect of the skull wasexposed with a longitudinal incision. Cannulas were inserted into thefourth ventricle (stereotaxic coordinates A/P −7.0, L±0.3 D/V −5.8),cerebral aqueduct (A/P −4.8 L÷D/V −2.6), or central canal (D/V −1.5).The cannulae were attached with sterile tubing to subcutaneouspositioned ALZET osmotic mini-pumps containing 25 μg/mL EGF (Becton40001) and/or 25 μg/mL FGF-2 (R&D Systems 233-FB). Pumps containingsterile saline plus 0.1% mouse albumin (Sigma A3134) were used ascontrols. The incisions were closed with dental cement.

[0264] Six days following surgery mice were injected with 0.15 mL BrdU(Sigma B5002); 18 mg/mL in 0.007% NaOH/0.1M PBS) every 2 hours for 8hours. They were killed 0.5 hours after the last injection with ananaesthetic overdose, and transcardially perfused with 10 mL of ice-coldsterile saline followed by 10 mL of ice-cold Bouin's fixative (5%glacial acetic acid, 9% formaldehyde, 70% picric acid). The cervicalspinal cord region was dissected out and post-fixed overnight at 4° C.in Bouin's post-fixative solution (9% formaldehyde, 70% picric acid).The following day the tissue was cryoprotected by immersion in 10%sucrose for 2 hours, 20% sucrose for 2 hours, and 30% sucrose overnight.The tissue was frozen in powdered dry ice, mounted in Tissue-Tek (Miles4583) at −18° C., and 30 μm serial sagittal sections were mounted ontogel-subbed glass slides. Each slide also contained one or more 30 μmcoronal sections through the lateral ventricles from the brain of thesame animal to serve as a positive control. Slides were kept at −80° C.until processed. Immunohistochemistry: Slides were rinsed in PBS 3×15minutes in 0.1M PBS at room temperature, hydrolyzed with 1N HCl for 60minutes at 37° C., rinsed for 3×15 minutes in 0.1M PBS at roomtemperature, placed in 6% H₂O₂ in methanol for 30 minutes at roomtemperature, rinsed for 3×15 minutes in 0.1M PBS at room temperature,and incubated in 10% normal horse serum (Sigma H-0146) in 0.1M PBS or 20minutes at room temperature. Slides were incubated overnight at roomtemperature in anti-BrdU monoclonal antibody (Becton 7580) that wasdiluted 1:50 in 0.1M PBS containing 1.5% normal horse serum and 0.3%Triton. The following day the slides were rinsed in PBS for 3×10 minutesin 0.1M PBS at room temperature, incubated with biotinylated horseanti-mouse IgG (Vector BA-2000) for 2 hours at room temperature, rinsedfor 3×15 minutes in 0.1M PBS at room temperature, incubated in ABCreagent (Vector PK-6100) for 2 hours at room temperature, rinsed for3×15 minutes in 0.1M PBS at room temperature, and developed with DABreagent for 2 to 4 minutes. The slides were coverslipped with AquaPolymount (Polysciences 18606). The number of BrdU positive cells wascounted per cervical spinal cord section. Some BrdU labelled cells werefound in the saline control sections. Treatment with either EGF or FGF-2resulted in a significant increase in the number of BrdU labelled cellsseen compared to control. The combination of EGF plus FGF-2 producedeven a greater amount of BrdU positive cells per section.

EXAMPLE 31 In Vivo Infusion of Growth Factors into Ventricles toIncrease Yield of Neural Stem Cells That Proliferate in vitro

[0265] EGF pumps were implanted as described in Example 27. Animals weresacrificed by cervical dislocation 6 days after the pump was implanted.Brains were removed and the stem cells isolated and counted as describedin Example 5.

[0266] Animals infused with EGF into the lateral ventricles for 6 daysprior to sacrifice and brain culturing had 4 times as many spheresforming after 9 days in vitro compared to control animals which receivedsaline pumps for the same 6 day period. Thus, infusing EGF into thelateral ventricles in vivo prior to removal and dissociation of neuraltissue, greatly increases the yield of stem cells which proliferate andform neurospheres in vitro. EGF and FGF can be infused into theventricles to further increase the yield of neural stem cells obtainablefrom the neural tissue. Neurospheres generated by this method are usedas a source of donor cells for later transplantation into degeneratedareas of human adult CNS. Neurospheres can also be proliferatedaccordingly from a patient's own CNS stem cells and transplanted backinto the patient.

EXAMPLE 32 In Vivo Modification of Neural Cells with bcl-2 Gene

[0267] A retroviral construct containing the human bcl-2 gene and theβ-galactosidase gene is microinjected into the adult mouse lateralventricle. A control mouse is injected with a retroviral constructcontaining only the β-galactosidase gene. One of the two progeny of eachof the constitutively proliferating subependymal cells of the adultlateral ventricle normally dies within a few hours after division. Thebcl-2 gene product prevents the programmed death of cells in severalother tissues. In the adult subependyma, single cells infected with boththe β-galactosidase and bcl-2 genes are marked by expression of boththese gene products. These cells are identified in brain tissue sliceswith antibodies specific to β-galactosidase and human Bcl-2.Proliferating infected subependymal cells so infected produce largernumbers of cells per clone relative to the control. Thus, Bcl-2 inducesthe survival of the one normally dying progeny of each division of aconstitutively proliferating adult subependymal cell. Moreover, thebcl-2 infected progeny migrate out into striatal and septal tissue toproduce new neurons and glia. This indicates that EGF and Bcl-2 act as asurvival factors for the normally dying progeny of constitutivelyproliferating adult subependymal cells which generate new neurons andglia in vivo.

EXAMPLE 33 In Vivo Modification of Neural Cells with NGF Gene

[0268] A retroviral construct containing the NGF gene is microinjectedusing the procedure described in Example 24 to infect the constitutivelyproliferating adult subependymal cells of the lateral ventricle. Thus,these cells are used to produce an endogenous growth factor in the adultbrain. Levels of NGF produced by the transfected cells are measureddirectly by radioimmunoassay and (from a direct functional assay) byrescue of basal forebrain cholinergic neurons in vivo after axotomyinjury in the model developed by Gage and collaborators (P.N.A.S.83:9231, 1986).

EXAMPLE 34 Generation of Dopamine Cells in the Striatum by theAdministration of a Composition Comprising Growth Factors to the LateralVentricle

[0269] Adult male CD₁ mice were anesthetized and placed in a stereotaxicapparatus. A cannula, attached to an ALZET minipump, was implanted intoa lateral ventricle of each animal. The minipumps were subcutaneouslyimplanted and were used to deliver (a) conditioned medium (from the ratB49 glial cell line, obtained from D Schubert, Salk Institute) plus bFGF(R&D Systems, 25 μg/ml) plus heparan sulfate (Sigma, 10 IU/ml) (CMF) or(b) EGF (Chiron, 25 μg/ml) plus bFGF (25 μg/ml) plus heparan sulfate (10IU/ml) plus 25% FBS (E+F+FBS) or (c) sterile saline solution (SAL) as acontrol, into the lateral ventricles. Once batch of animals wassacrificed one day after completion of the delivery regimen and theothers were sacrificed twenty days later. The subventricular zones(SVZs) of these mice were dissected out, separating the cannulated, andtherefore treated, side from the non-cannulated control sides. Thesubstantia nigra (SN) region of these mice were also recovered. TotalRNA was extracted from these tissues using the guanidium thiocyanateacid phenol method [Chomzynski and Sacchi, Annal. Biochem. 162: 156-159,(1987)]. The RNA was then reverse transcribed to produce cDNA. ThesecDNAs were subject to PCR using primers designed to bracket a 254nucleotide region of the TH messenger RNA (mRNA) and thermal cyclingconditions favoring quantitative amplification. The PCR products wereelectrophoresed on a 2% agarose gel and then capillary blotted onto apositively charged nylon membrane. Radioactively labelled cDNA probe toTH was hybridized to the filter and detected by autoradiography. Theautoradiograph was scanned and analyzed by densitometry to obtainrelative levels of mRNA for TH in the SVZs of the cannulated sides inresponse to the treatments in the non-cannulated control SVZs and in theSN. In animals analyzed one day after treatment, the administration ofE+F+FBS produced an eleven-fold increase in the level of TH mRNA in theSVZ compared to that observed in response to CMF, which in turn was morethan twice the level seen with SAL. Twenty one days after treatment, theamount of TH mRNA detected in response to treatment with E+F+FBS wasapproximately the same as that detected after one day, while CMF and SALtreated SVZs had TH mRNA levels which were below detectable limits andwere indistinguishable from the non-cannulated SVZ controls. Under alltreatments, the SN had measurable amounts of TH mRNA.

EXAMPLE 35 Detection of Dopaminergic Cells in Striatal Tissue Using DualLabeling

[0270] Male CD₁ mice (Charles River, approximately 4 to 6 weeks old)were given intraperitoneal injections of BrdU (Sigma, 120 mg/kg) at 2hour intervals over a 24 hour period, in order to label mitoticallyactive cells. A cannula attached to an ALZET minipump was then implantedunilaterally into a lateral ventricle of each animal in order to delivercompositions a-c (CMF, E+F+FBS, or sterile saline) described in Example34.

[0271] Animals were sacrificed 24 hours after the administration ofgrowth factors using a lethal dose of pentobarbital anesthetic. Theanimals were then perfused through the heart with 10 ml of ice could 4%paraforinaldehyde solution. The brains were removed and tissue in theregion extending from the olfactory bulb to the third ventricle,including the striatum, was dissected out and stored overnight at 4° C.in a 30% sucrose/4% paraformaldehyde solution. The tissue was thenfrozen on dry ice and kept at −70° C. until processed. 30 μm coronalsections were cut using a cryostat and the sections were placed in 12well porcelain dishes, to which 400 μl of PBS had been added. Sectionswere rinsed with fresh PBS and incubated overnight with the followingprimary antibodies: anti-TH (rabbit polyclonal, 1:1000, Eugene TechInternational Inc.; or 1:100, Pel-freeze) and mouse anti-BrdU (1:55,Amersham), prepared in PBS/10% normal goat serum/0.3 Triton X-100.Following three rinses in PBS, goat anti-rabbit rhodamine and goatanti-mouse fluorescein (Jackson) were applied in PBS for 50 minutes atroom temperature. Sections were then washed three times (10 minuteseach) in PBS, placed on glass slides, dried and then coverslipped usingFluorsave (Calbiochem #345789).

[0272] The location of dopaminergic neurons was determined by mappingthe location of TH-immunoreactive (TH+) cells, or TH+ and BrdU+ cells inrelation to the ventricles. In response to saline injections made intothe lateral ventricles, the normal population of TH+ fibers weredetected in the striatum but no TH+ cell bodies were detected in thisregion. CMF treatment resulted in the detection of TH+ cell bodies, inaddition to the normal population of TH+ fibers, in the striatum and inthe region of the third ventricle. E+F+ FBS treatment had the mostprofound effect resulting in the detection of the most TH+ cell bodies.Several of the TH+ cell bodies were also BrdU positive.

EXAMPLE 36 Rat Model of Parkinson's Disease Measures the Effects of InVivo Administration of Growth Factors

[0273] The 6-OHDA lesion rat model of Parkinson's disease is used tomeasure the effects of administering various combinations of growthfactors to the lateral ventricle. Unilateral 6-OHDA lesions areperformed in the rat model and rotation behavior is observed. Minipumpsare subcutaneously implanted into the animals as described in Example34. EGF (Chiron, 25 μg/ml) plus bFGF (25 g/ml) plus heparan sulfate (10IU/ml) plus 25% FBS is continuously administered to the lateralventricle. Saline is administered to control animals. The ability toovercome the rotational bias produced by the unilateral 6-OHDA lesionsis observed.

EXAMPLE 37 Screening of Drugs or Other Biological Agents for Effects onMultipotent Neural Stem Cells and Neural Stem Cell Progeny

[0274] A. Effects of BDNF on Neuronal and Glial Cell Differentiation andSurvival

[0275] Precursor cells were propagated as described in Example 4 anddifferentiated using Paradigm 3 described in Example 7. At the time ofplating the EGF-generated cells, BDNF was added at a concentration of 10ng/ml. At 3, 7, 14, and 21 days in vitro (DIV), cells were processed forindirect immunocytochemistry. BrdU labeling was used to monitorproliferation of the precursor cells. The effects of BDNF on neurons,oligodendrocytes and astrocytes were assayed by probing the cultureswith antibodies that recognize antigens found on neurons (MAP-2, NSE,NF), oligodendrocytes (O4, GalC, MBP) or astrocytes (GFAP). Cellsurvival was determined by counting the number of immunoreactive cellsat each time point and morphological observations were made. BDNFsignificantly increased the differentiation and survival of neurons overthe number observed under control conditions. Astrocyte andoligodendrocyte numbers were not significantly altered from controlvalues.

[0276] B. Effects of BDNF on the Differentiation of Neural Phenotypes

[0277] Cells treated with BDNF according to the methods described inPart A were probed with antibodies that recognize neural transmitters orenzymes involved in the synthesis of neural transmitters. These includedTH, ChAT, substance P, GABA, somatostatin, and glutamate. In bothcontrol and BDNF-treated culture conditions, neurons tested positive forthe presence of substance P and GABA. As well as an increase in numbers,neurons grown in BDNF showed a dramatic increase in neurite extensionand branching when compared with control examples.

[0278] C. Identification of Growth-Factor Responsive Cells

[0279] Cells that are responsive to growth factor treatment wereidentified by differentiating the EGF-generated progeny as described inExample 7, paradigm 3 and at 1 DIV adding approximately 100 ng/ml ofBDNF. At 1, 3, 6, 12 and 24 hours after the addition of BDNF the cellswere fixed and processed for dual label immunocytochemistry. Antibodiesthat recognize neurons (MAP-2, NSE, N), oligodendrocytes (04, GalC, MBP)or astrocytes (GFAP) were used in combination with an antibody thatrecognizes c-fos and/or other immediate early genes. Exposure to BDNFresults in a selective increase in the expression of c-fos in neuronalcells.

[0280] D. Effects of BDNF on the Expression of Markers and RegulatoryFactors During Proliferation and Differentiation

[0281] Cells treated with BDNF according to the methods described inPart A are processed for analysis of the expression of FGF-RI, asdescribed in Example 39 or other markers and regulatory factors, asdescribed in Example 40.

[0282] E. Effects of BDNF administration During Differentiation on theElectrophysiological Properties of Neurons

[0283] Neurons treated with BDNF during differentiation, according tothe methods described in Part A, are processed for the determination oftheir electrophysiological properties, as described in Example 41.

[0284] F. Effects of Chlorpromazine on the Proliferation,Differentiation, and Survival of Growth Factor Generated Stem CellProgeny

[0285] Chlorpromazine, a drug widely used in the treatment ofpsychiatric illness, is used in concentrations ranging from 10 ng/ml to1000 ng/ml in place of BDNF in Examples 7A to 7E above. The effects ofthe drug at various concentrations on stem cell proliferation and onstem cell progeny differentiation and survival is monitored. Alterationsin gene expression and electrophysiological properties of differentiatedneurons are determined.

EXAMPLE 38 Stem Cell Proliferation Assay

[0286] Primary cells were obtained from E14 mice and prepared asdetailed in Examples 1 and 4. Either EGF, EGF and FGF or EGF and BMP-2were added to complete medium at a concentration of 20 ng/ml of eachgrowth factor, with the exception of BMP-2 which was added at aconcentration of 10 ng/ml. Cells were diluted with one of the preparedgrowth factor-containing media to a concentration of 25,000 cells/ml.200 μl of the cell/medium combination were pipetted into each well of a96-well place (Nuclon) with no substrate pretreatment. Cells wereincubated under the same conditions as outlined in Example 4.

[0287] After 8-10 DIV the number of neurospheres was counted and theresults tabulated As Cells grown in a combination of EGF and FGFproduced significantly more neurospheres than cells grown in thepresence of EGF alone. The combination of EGF and BMP-2 inhibitedneurosphere development.

EXAMPLE 39 Comparison of Receptor and Growth Factor Expression inUndifferentiated vs. Differentiated Stem Cell-Derived Progeny by ReverseTranscription-Polymerase Chain Reaction (RT-PCR)

[0288] Neurospheres were generated as described in Example 4, and somewee differentiated as per Paradigm 1, Example 7. RNA from eitherundifferentiated or differentiated neurospheres was isolated accordingto the guanidinium thiocyanate acid phenol procedure of Chomzynski andSacchi—Anal. Biochem. 162: 156-159 1987)]. Complementary DNA (cDNA) wassynthesized from total RNA using reverse transcriptase primed with oligodT. Gene-specific primers were designed and synthesized and theseprimers were used in PCR to amplify cDNAs for different growth factorsand growth factor receptors. Amplified material was run on agarose gelsalongside molecular weight markers to ensure that PCR products were ofthe expected size, while the identity of PCR fragments was confirmed byrestriction enzyme analysis and by sequencing [Arcellana-Panlilio,Methods Enzymol. 225: 303-328 (1993)]. An ethidium-stained agarose gelvisualized via UV transillumination showed the detection of three growthfactor receptor transcripts, namely EGF-R, FGF-R, and LIF-R, inundifferentiated and differentiated stem cell-derived progeny. Table Ilists the primer sets analyzed and the results of undifferentiated anddifferentiated cells. TABLE I Primer Sets Analyzed UndifferentiatedDifferentiated Cells Cells Actin + + NGF + nd EGFr^(m) + + bFGFr + +LIFr^(m) + + tyrosine hydroxylase + + choline acetyltransferase^(m) nd +cholecystokinin^(m) nd − enkephalin^(m) nd + tyrosine kinase-rA + +tyrosine kinase-rB + +++++ tyrosine kinase-rC + +

EXAMPLE 40 Isolation of Novel Markers and Regulatory Factors Involved inNeural Stem Cell Proliferation and Differentiation

[0289] Neurospheres are generated as described in Example 4 using CNStissue from CD, albino mice (Charles River). Some of these neurospheresare allowed to differentiate according to the rapid differentiationparadigm of Example 7 producing cultures enriched in neurons,astrocytes, and oligodendrocytes. Total RNA is extracted from theundifferentiated neurospheres as well as the differentiated cellcultures using the guanidinium thiocyanate acid phenol method referredto in Example 39. Messenger RNA (mRNA) is isolated by exploiting theaffinity of its poly A tract to stretches of either U's or T's. Reversetranscription of the mRNA produced cDNA, is then used to make primarylibraries in either plasmid [Rothstein et al., Methods in Enzymology225:587-610 (1993)] or lambda phage vectors. To isolate cDNAs that arespecific to either undifferentiated or differentiated stem cell derivedprogeny, cDNA from one is hybridized to RNA from the other, and viceversa. The unhybridized, and thus culture type-specific, cDNAs in eachcase are then used to construct subtracted libraries [Lopez-Fernandezand del Mazo, Biotechniques 15(4):654-658 (1993)], or used to screen theprimary libraries.

[0290] Stem cell-derived undifferentiated cell specific anddifferentiated cell specific cDNA libraries provide a source of clonesfor novel markers and regulatory factors involved in CNS stem cellproliferation and differentiation. Specific cDNAs are studied bysequencing analysis to detect specific sequence motifs as clues toidentity or function, and database searching for homologies to knowntranscripts. Using cDNAs in a hybridization to various RNA sampleselectrophoresed on an agarose-formaldehyde gel and transferred to anylon membrane, allows the estimation of size, relative abundance, andspecificity of transcripts. All or portions of cDNA sequences are usedto screen other libraries in order to obtain either complete mRNAsequences or genomic sequence information. Antibodies directed againstfusion proteins generated from specific cDNAs are used to detectproteins specific to a particular cell population, either byimmunocytochemistry or by Western Blot analysis. Specific gene sequencesare used to isolate proteins that interact with putative regulatoryelements that control gene expression. These regulatory elements arethen used to drive the expression of an exogenous gene, such asbeta-galactosidase.

EXAMPLE 41 Electrophysiological Analysis of Neurons Generated FromGrowth Factor-Responsive Stem Cells and Exposed to a Biological Agent

[0291] Neurospheres were generated as described in Example 4.Neurospheres were dissociated using the technique described in paradigm2, Example 7. The clonally derived cells were plated at low density anddifferentiated in the presence of bFGF. The electrophysiologicalproperties of cells with the morphological appearance of neurons weredetermined as described as described by Vescovi et al. [Neuron, 11:951-966 (1993)]. Under whole cell current clamp, the mean restingpotential and input resistance were −62±9 mV and 372±MΩ. Rectangularsuprathreshold current steps, (−100 pA) elicited regenerative potentialresponses in which the amplitude and time course were stimulusdependent. After the completion of electrophysiological experiments, thecell morphology was visualized by intracellular excitation of5-carboxyfluorescein.

EXAMPLE 42 Screening for the Effects of Drugs or Other Biological Agentson Growth Factor-Responsive Stem Cell Progeny Generated From TissueObtained From a Patient with a Neurological Disorder

[0292] The effects of BDNF on the EGF-responsive stem cell progenygenerated from CNS tissue obtained at biopsy from a patient withHuntington's disease is determined using the methods outlined in Example7, A to E. BDNF is a potent differentiation factor for GABAergic neuronsand promotes extensive neuronal outgrowth. Huntington's Disease ischaracterized by the loss of GABAergic neurons (amongst others) from thestriatum.

EXAMPLE 43 Assay of Striatum-Derived Neurosphere Proliferation inResponse to Various Combinations of Proliferative and Regulatory Factors

[0293] Paradigm 1:

[0294] Primary striatal cells prepared as outlined in Example 1 weresuspended in Complete Medium, without growth factors, plated in 96 wellplates (Nunclon) and incubated as described in Example 4. Following aone hour incubation period, a specific proliferative factor, or acombination of proliferative factors including EGF, or bFGF (recombinanthuman bFGF: R & D Systems), or a combination of EGF and bFGF, or EGFplus FGF plus heparan sulfate (Sigma), or bFGF plus heparan sulfate madeup in Complete Medium at a concentration of 20 ng/ml for each of thegrowth factors and 2 μg/ml for heparan sulfate), was added to each wellof the plate.

[0295] Activin, BMP-2, TGF-β, IL-2, IL-6, IL-8, MIP-1∂, MIP-1β, MIP-2(all obtained from Chiron Corp.), TNFα, NGF (Sigma), PDGF (R&D Systems),EGF and CNTF (R. Dunn and P. Richardson, McGill University) were made upin separate flasks of compete medium to a final concentration of 0.2μg/ml. Retinoic acid (Sigma) was added at a concentration of 10⁻⁶ M. 10μl of one of these regulatory factor-containing solutions was added toeach proliferative factor-containing well of the 96 well plates. Controlwells, containing only proliferative factors, were also prepared.

[0296] In another set of experiments, the neurosphere inducingproperties of each of these regulatory factors was tested by growingcells in their presence, in proliferative factor-free Complete Medium.None of these regulatory factors, with the exception of EGF, when usedin the absence of a proliferation-inducing factor such as EGF or FGF,has an effect on neural stem cell proliferation.

[0297] The activin, BMP-2, TGF-β, IL-2, IL-6, IL-8, MIP-1∂, MIP-1β,MIP-2, TNFα and EGF additions were repeated every second day, CNTF whichwas added each day and retinoic acid, NGF and PDGF were added only once,at the beginning of the experiment. The cells were incubated for aperiod of 10-12 days. The number of neurospheres in each well wascounted and the resulting counts tabulated using Cricket Graph III.Other relevant information regarding sphere size and shape were alsonoted.

[0298] In general, bFGF had a greater proliferative effect than EGF onthe numbers of neurospheres generated per well. In the presence of 20ng/ml EGF, approximately 29 neurospheres per well were generated. In thepresence of bFGF, approximately 70 neurospheres were generated. However,in bFGF alone, the neurospheres were only about 20% of the size of thosegenerated in the presence of EGF. The combination of EGF and bFGFproduces significantly more neurospheres than does EGF alone, but fewerthan seen with bFGF alone. The neurospheres are larger than those seenin bFGF alone, approximating those seen in EGF. In the case of bFGFgenerated spheres, the addition of heparan sulfate increased the size ofthe spheres to about 70% of the size of those which occur in response toEGF. These data suggest that EGF and FGF have different actions withrespect to the induction of stem cell mitogenesis.

[0299] The effects of the regulatory factors added to the proliferativefactor-containing wells are summarized in Table II. In general, the TGFβfamily, interleukins, macrophage-inhibitory proteins, PDGF, TNFα,retinoic acid (10⁻⁶M) and CNTF significantly reduced the numbers ofneurospheres generated in all of the proliferative factors orcombinations of proliferative factors tested. BMP-2 (at a dose of 10ng/ml), completely abolished neurosphere proliferation in response toEGF. EGF and heparan sulfate both greatly increased the size of theneurospheres formed in response to bFGF (about 400%). TABLE IIPROLIFERATIVE FACTORS EGF + bFGF + bFGF + EGF bFGF EGF+ bFGF HeparanHeparan REGULATORY FACTORS # size # size # size # size # size TGFβFamily ♦ −57% - −57% - −34% -- −55% - −20% - BMP-2 −100% n/a −5% = +16%-- −3% - +10% -- Interleukins −21% = −23% = −37% - −28% = −39% - MIPFamily −25% = −6% = −32% - −22% = −33% - NGF −10% = 0% = −30% = +5% =−48% = PDGF −1.5% = −4% = −26% = −10% = −27% = TNFα −17% = −17% = −41% =−21% = −37% = 10⁻⁶M Retinoic Acid −8% -- −61% - −31% -- −65% -- −45% --CNTF −23% - −77% ˜ −81% -- −81% - −84% -- EGF - −14% ++ - −17% = -Heparan Sulfate 0% = 0% ++ 0% =

[0300] Antisense and sense experiments were carried out using thefollowing oligodeoxynucleotides (all sequences written 5′ to 3′): EGFSense strand: GAGATGCGACCCTCAGGGAC receptor: Antisense strand:GTCCCTGAGGGTCGCATCTC EGF: Sense strand: TAAATAAAAGATGCCCTGG Antisensestrand: CCAGGGCATCTTTTATTTA

[0301] Each oligodeoxynucleotide was brought up and diluted in ddH₂O andkept at −20° C. Each well of the 96 well plates received 10 μl ofoligodeoxynucleotide to give a final concentration of either 1, 2, 3, 4,5, 10 or 25 μM. Oligodeoxynucleotides were added every 24 hours. The EGFreceptor (EGFr) and EGF oligodeoxynucleotides were applied to culturesgrown in bFGF (20 ng/ml), and EGFr oligodeoxynucleotides were applied tocultures grown in EGF (20 ng/ml). Cells were incubated at 37° C., in a5% CO₂ 100% humidity incubator. After a period of 10 to 12 days, thenumber of neurospheres per well was counted and tabulated. Aconcentration of 3 μM of antisense oligodeoxynucleotides produced a 50%reduction in the number of neurospheres generated per well, whereas thesense oligodeoxynucleotides had no effect on the number of neurospheresgenerated in response to EGF and FGF. Both the sense and antisenseoligodeoxynucleotides were toxic to cells when 10 μM or higherconcentrations were used.

[0302] Similar experiments can be performed using the followingoligonucleotides: FGF Sense strand: GAACTGGGATGTGGGGCTGG receptor:Antisense strand: CCAGCCCCACATCCCAGTTC FGF: Sense strand:GCCAGCGGCATCACCTCG Antisense strand: CGAGGTGATGCCGCTGGC

[0303] The FGF receptor (FGFr) and FGF oligodeoxynucleotides are appliedto cultures grown in EGF, and FGFr oligodeoxynucleotides are applied tocultures grown in bFGF.

[0304] Paradigm 3:

[0305] Embryonic tissue is prepared as outlined in Example 1 and platedinto 96 well plates. Complete Medium, containing 20 ng/ml of either EGFof bFGF is added to each well. 10 μl of diluted phorbol 12-myristate 13acetate (PMA) is added once, at the beginning of the experiment, to eachwell of the 96 well plates, using an Eppendorf repeat pipetter with a500 μl tip to give a final concentration of either 10, 20, 40, 100 or200 μg/ml. Cells are incubated at 37° C. in a 5% CO₂ 100% humidityincubator. After a period of 10 to 12 days the number of neurospheresper well is counted and tabulated.

[0306] Paradigm 4:

[0307] Embryonic tissue is prepared as outlined in Example 1 and platedinto 96 well plates. 10 μl of diluted staurosporine is added to eachwell of a 96 well plate, using an Eppendorf repeat pipetter with a 500μl tip to give a final concentration of either 10, 1, 0.1, or 0.001 μMof staurosporine. Cells are incubated at 37° C., in a 5% CO₂ 100%humidity incubator. After a period of 10 to 12 days, the number ofneurospheres per well is counted and tabulated.

EXAMPLE 44 Adult Spinal Cord Stem Cell Proliferation—in vitro Responsesto Specific Biological Factors or Combinations of Factors

[0308] Spinal cord tissue was removed from 6 week to 6 month old mice,as follows: cervical tissue was removed from the vertebral column regionrostral to the first rib; thoracic spinal tissue was obtained from theregion caudal to the first rib and approximately 5 mm rostral to thelast rib; lumbar-sacral tissue constituted the remainder of the spinalcord. The dissected tissue was washed in regular artificialcerebrospinal fluid (aCSF), chopped into small pieces and then placedinto a spinner flask containing oxygenated aCSF with high Mg²⁺ and lowCa²⁺ and a trypsin/hyaluronidase and kynurenic acid enzyme mix tofacilitate dissociation of the tissue. The tissue was oxygenated,stirred and heated at 30° C. for 1½ hours, then transferred to a vialfor treatment with a trypsin inhibitor in media solution(DMEM/12/hormone mix). The tissue was triturated 25-50 times with a firenarrow polished pipette. The dissociated cells were centrifuged at 400r.p.m. for 5 minutes and then resuspended in fresh media solution. Cellswere plated in 35 mm dishes (Costar) and allowed to settle. Most of themedia was aspirated and fresh media was added. EGF alone, or EGF andbFGF were added to some of the dishes to give a final concentration of20 ng/ml each, and bFGF (20 ng/ml) was added, together with 2 μg/ml ofheparan sulfate, to the remainder of the dishes. The cells wereincubated in 5% CO₂, 100% humidity, at 37° C. for 10-14 days. Thenumbers of neurospheres generated per well were counted and the resultstabulated. EGF alone resulted in the generation of no neurospheres fromany of the spinal cord regions. In the presence of EGF plus bFGF,neurospheres were generated from all regions of the spinal cord, inparticular the lumbar sacral region. The combinations of EGF+FGF andFGF+heparan sulfate produced similar numbers of spheres in the cervicalregion, whereas the combination of bFGF plus heparan sulfate resulted infewer neurospheres from the thoracic and lumbar regions.

EXAMPLE 45 Transplantation of Multipotent Neural Stem Cell Progeny inAnimal Models

[0309] I. Transplantation Procedure

[0310] 1. Neurosphere Preparation

[0311] Neural tissue was obtained from normal embryonic or adult CD1mice and from embryonic or adult Rosa 26 mice (transgenic animalsderived from C57/BL/6 mice, which express the β-galactosidase gene inall cells, thus allowing the transplanted cells to be easily detected inhost tissue). Neurospheres were generated using the procedures describedin Examples 1-5, passaged 2 to 8 times (see Example 6), and maintainedin culture for 6-10 days after the last passage.

[0312] 2. Labeling and Preparation of Neural Stem Cell Progeny

[0313] 16 hours prior to transplantation, neurospheres derived fromembryonic and adult tissue were labeled with BrdU by adding BrdU to themedia for a total concentration of 1 μM and/or with fluorescent latexbeads (Polysciences; 1:100 dilution of 0.75μm beads). Neurospheres weredetached from the substrate by gentle shaking, poured into 50 mlcentrifuge tubes and spun down (5 minutes, 400 r.p.m., 15° C., no brake)to remove the proliferation-inducing media used for the proliferationculture. The neurospheres derived from embryonic tissue were then washedtwice in Hank's buffered salt solution (HBSS), resuspended in 2 ml HBSSand dissociated by trituration (spheres drawn into a fire-polishedpasteur pipette 40×). The neurospheres derived from adult tissue weretrypsinized (0.05% in EDTA media; 5-10 min) and then a trypsin inhibitor(ovomucoid; 0.7-1.0 mg/ml in media) was added. The tubes were swirledand the neurospheres were recentrifuged (400 r.p.m., 15° C., no brake).Cells were resuspended in 2 ml media (DMEM F12/hormone mix) anddissociated by mechanical trituration (25×).

[0314] Live and dead cells obtained from neurospheres derived fromembryonic and adult tissue were counted prior to being centrifuged toremove dead cells (10 min., 400 r.p.m., 15° C., no brake). The livecells were resuspended to appropriate cell density (1-50×10⁶ cells/ml).The cells were recounted to determine the number of live and dead cellsand cell viability was calculated. The cells were then transferred to amicrocentrifuge tube for storage on ice prior to transplantation. Whenready for use, cells were resuspended prior to each cell injection bydrawing cells into an eppendorf pipette tip (200 or 1000 μl).

[0315] 3. Transplantation of Neural Stem Cell Progeny

[0316] The donor neural stem cell progeny were transplanted intoselected sites in the brain of normal, healthy neonate or adult CD1 orC57BL/6 mice or adult Wistar or Sprague-Dawley rats. In some cases,embryonic cells from CD1 mice received in vitro gene transfer proceduresprior to transplantation of the cells. The host animals wereanaesthetized with sodium pentobarbital (65 mg/Kg) and placed into astereotaxic apparatus. A skin incision was made to expose the surface ofthe skull or vertebrae. Injection sites were located using stereotaxiccoordinates to locate the desired site. Burr holes were drilled in theskull and vertebrae at the coordinate sites. A 5 μl syringe was housedon a syringe pump and attached to a stainless steel cannula (30-31gauge) via a short length of polyethylene tubing. A small air bubble andthen 4-5 μl of the desired cell suspension was drawn into the cannula.The cannula was lowered to the desired location and 1-3 μl of the cellsuspension was injected at a speed of 0.1-0.5 μl/min. Animals thatreceived xenografts or allograft were treated with 0.1 mg/ml cyclosporinA in the drinking water to reduce the risk of tissue rejection.

[0317] 4. Analysis of Transplanted Neural Stem Cell Progeny

[0318] The animals were allowed to survive for 2-12 weeks prior tosacrifice. At a specified time after transplantation, animals wereperfused transcardially for aldehyde fixation of the brain and spinalcord tissue. A low-high pH perfusion protocol was used (Sloviter &Nilayer, (1987) Brain Res. Vol. 330:358-363). After perfusion, brainsand spinal cords were removed, post-fixed, and then cryoprotected insucrose/PBS for cutting in a cryostat. Sections of tissue (10 μM) werecut and mounted on microscope slides directly in a sequential way sothat adjacent sections could be examined with different anatomicalprotocols.

[0319] Survival of transplanted cells labeled with fluorescent beadswere identified by the localization of fluorescent beads within the cellcytoplasm. BrdU labeled cells (cells that had incorporated BrdU intotheir DNA during cell division in culture prior to transplantation) wereidentified using antibodies against BrdU (1:250-500;Monoclonal-Sera-lab; Polyclonal-Accurate Chem. & Sci). Antibodiesagainst GFAP (1:250 Monoclona-Boehringer, Polyclonal-BTI), or NeuN(1:250-500; Monoclonal-R. J. Mullen) were then used to identify thedifferentiation of the transplanted cells. Cell transplants derived fromtransgenic animals expressing β-galactosidase were histochemicallyanalyzed using methodology described by Turner and Cepko (1987) Nature328:131-136)and by immunohistochemical staining. For Rosa 26 cells,antibodies against β-galactosidase were used to identify thetransplanted cells and antibodies to NeuN were used to identify cellsthat had differentiated into neurons. Human cells were identified withHLA antibodies (1:250, Monoclonal-Sera-labs). Antibodies were incubatedwith the tissue samples and detected using standard immunohistochemicalprotocols.

[0320] The results obtained from the animal models described below aresummarized in Tables II-V.

[0321] A. Model of Huntington's Disease

[0322] Rats were anesthetized with nembutal (25 mg/kg i.p) and injectedwith atropine sulfate (2 mg/kg i.p.). Animals sustained an ibotenatelesion of the striatum, stimulating Huntington's Disease in the animals.7 days after the lesion, the animals received an injection of cellsprepared as in Examples 1-5 under stereotaxic control. Injections weremade to the lesioned area via a 21-gauge cannula fitted with a tefloncatheter to a microinjector. Injected cells were labelled withfluorescein-labelled microspheres. Animals were given behavioral testsbefore the lesion, after the lesion, and at various intervals after thetransplant to determine the functionality of the grafted cells atvarious postoperative time points, then killed and perfusedtranscardially with 4% buffered paraformaldehyde, 0.1% glutaraldehydeand 5˜°a sucrose solution at 4 C. The brains were frozen in liquidnitrogen and stored at −20° C. until use. Brains sections were sliced to26 μm on a cryostat, fixed in 4% paraformaldehyde and stained using theM6 monoclonal antibody to stain for mouse neurons, and then reacted witha secondary anti-rat fluorescein-conjugated antibody. Neuronal and glialphenotype was identified by dual labeling of the cells with antibody toNSE and GFAP.

[0323] B. Parkinson's Disease

[0324] Two animal models of Parkinson's Disease were used. In the firstmodel, unilateral dopamine neurons of the substantia nigra were lesionedby the stereotaxic administration of 6-OHDA into the substantia nigra inadult CD 1 (14 μg) and C57BL/6 mice (1 μg), and Wistar rats (16 μg).Mice were pretreated with desipramine (25 mg/Kg i.p.) and rats werepretreated with pargyline (50 mg/Kg i.p.) both of which prevent theaction of 6-OHDA on noradrenergic neurons and allow the selectivedestruction of dopaminergic neurons. In one series of experiments,multipotent neural stem cell progeny obtained from embryonic Rosa 26mice, were prepared using the procedures described in Examples 1 and 4.The neural stem cell progeny were labeled, prepared, and transplantedinto the striatum of the lesioned C57BL/6 mice using the methodsdescribed above in this Example.

[0325] In a second series of experiments, the cells were administered tothe same regions in the brains of adult 6-OHDA Wister rats. In a thirdseries of experiments, proliferated fetal human cells (prepared asoutlined in Example 9), were transplanted into the striatum of the600HDA lesioned CD1 mice. After a survival period of 2 weeks, the hostanimals were sacrificed and their brains removed. The brain tissue wastreated and analyzed as described above.

[0326] The second animal model used was the adult mutant Weaver mice(Jackson Labs, 3½ months), in which approximately 70% of thedopaminergic neurons of the substantia nigra are lost by the age of 3months. Animals were anaesthetized and the proliferated progeny ofmultipotent neural stem cells derived from embryonic Rosa 26 mice wereinjected into the striatal region of the animals according to themethods described above. The animals were allowed to survive for 15 daysprior to sacrifice and analysis of striatal tissue.

[0327] C. Cardiac Arrest

[0328] Transient forebrain ischemia was induced in adult Wistar rats bycombining bilateral carotid occlusion with hypovolemic hypotension(Smith et al. (1984) Acta Neurol Scand 69:385401). These procedureslesion the CA1 hippocampal pyramidal cells which is typical of damageobserved in humans following cardiac arrest and the cause of severememory and cognitive deficits. The progeny of proliferated multipotentneural stem cells, derived from embryonic Rosa 26 mice, were prepared asdescribed above and transplanted into the striatal region of theischemia lesioned rats. After 8 days, the animals were sacrificed andtheir brains were removed and analyzed. β-gal positive cells, indicatingsurviving cells from the Rosa 26 donor) were detected in the lesionedhippocampal region. In addition, double labeled β-gal/NeuN⁺ cells werefound indicating that transplanted cells had differentiated intoneurons.

[0329] D. Stroke

[0330] Occlusion of the carotid arteries precipitates the occurrence ofischemic damage similar to that which occurs during stroke. Adult Wistarrats, in which the middle cerebral artery has been occluded to producesymptomatic lesions in the caudal striatum and parietal cortex, haveneural stem cell progeny implanted into the lesioned areas. After asurvival period, the animals are tested for behavioral improvements andare then sacrificed and their brains analyzed.

[0331] E. Epilepsy

[0332] Implantation of an electrode into the amygdala is used to kindlethe brain, inducing epileptic episodes and other symptoms of epilepsy.Neural stem cell progeny are transplanted into the hippocampal regionThe animals are later tested for epileptic episodes and then sacrificedfor analysis of the grafted tissue.

[0333] F. Alzheimer's Disease

[0334] Cognitive impairment is induced in rats and mice by ibotenic acidlesions of the nucleus basalis, or old animals, exhibiting signs ofdementia, are used. Neural stem cell progeny are transplanted into thefrontal cortex, medial septal nucleus and the nucleus of the diagonalband of the brains of the animals. After a survival period, the animalsare tested for cognitive ability and are then sacrificed to allowanalysis of brain tissue.

[0335] G. Spinal Cord Injury and Disease

[0336] Spasticity is a debilitating motor disorder that is a commonconsequence of disorders such as spinal cord injury, MS, and cerebralpalsy. Transection of the spinal cord is used to produce muscularparalysis and is followed by the development of spasticity, which ischaracterized by debilitating hyperactive tendon reflexes, clonus andmuscle spasms. Neural stem cell progeny are prepared and aretransplanted into the lumbar lateral funiculus. After a survival period,the animals are examined for improvement in motor control and are thensacrificed to allow for analysis of spinal tissue. TABLE III DONOR CELLTRANSPLANT BrdU/ SOURCE HOST REGION BrdU BrdU/GFAP NeuN Embryonic CD1Neonate CD1 striatum + + + Mouse Mouse frontal cortex + + + Adult CD1striatum + + + Mouse hippocampus + + + frontal cortex + + + parietalcortex + + + MS/NDB + + + Adult Wistar Rat spinal cord + + +hippocampus + + + parietal cortex + + Adult CD1 Mouse Adult CD1striatum + + Mouse hippocampus + frontal cortex + Adult Wistar Ratspinal cord +

[0337] TABLE IV Donor Cell Transplant BrdU/ Source HOST Region β-GalBrdU GFAP Embryonic Adult CD1 hippocampus + + + CD1 Mouse Mouse frontalcortex + + + in vitro gene parietal cortex + + + transfer striatum + + +MS/NDB + + + Embryonic Adult DC1 striatum + + + Rosa Mouse parietalcortex + + + MS/NDB + + + Adult Rosa 26 Adult hippocampus + C57/BL/6frontal cortex + Mouse MS/NOB +

[0338] TABLE V DONOR CELL SOURCE HOST β-Gal BrdU/GFAP Embryonic Adult6-OHDA lesioned + + Rosa 26 Mouse C57BL/6 mouse (striatal injections)Adult 6-OHDA lesioned Wistar + + rat (striatal injections) EmbryonicAdult Mutant Weaver Mouse + + Rosa 26 Mouse (striatal injections)

[0339] All references, patents, and patent applications cited herein areincorporated herein by reference.

We claim:
 1. A method of proliferating a culture of undifferentiatedneural cells containing one or more multipotent neural stem cells,wherein said undifferentiated neural cells are nestin⁺ and are glialfibrillary acidic protein (GFAP)⁻; neurofilament (NF)⁻; neuron specificenolase (NSE)⁻; and myelin basic protein (MBP)⁻; and not nestin, themethod comprising culturing the undifferentiated neural cells in aculture medium containing at least one proliferation-inducing growthfactor.
 2. The method of claim 1, wherein the proliferation-inducinggrowth factor is selected from the group consisting of epidermal growthfactor, amphiregulin, acidic fibroblast growth factor, basic fibroblastgrowth factor, transforming growth factor alpha, and combinationsthereof.
 3. The method of claim 1, wherein said culture medium lacksserum.
 4. The method of claim 1, wherein the undifferentiated neuralcells are human cells.
 5. The method of claim 1, wherein theundifferentiated neural cells are cultured in a suspension culture. 6.The method of claim 1, wherein the undifferentiated neural cells arecultured in an adherent culture.
 7. A method of proliferating a cultureof undifferentiated neural cells containing one or more multipotentneural stem cells, wherein said undifferentiated neural cells that stainpositive for nestin and said undifferentiated neural cells lackdifferentiated neural cells that do not stain positive for nestin andthat stain positive for the differentiated neural cell markersneurofilament, glial fibrillary acidic protein, neuron specific enolase,and myelin basic protein, the method comprising culturing theundifferentiated neural cells in a culture medium containing at leastone proliferation-inducing growth factor.
 8. The method of claim 7,wherein the proliferation-inducing growth factor is selected from thegroup consisting of epidermal growth factor, amphiregulin, acidicfibroblast growth factor, basic fibroblast growth factor, transforminggrowth factor alpha, and combinations thereof.
 9. The method of claim 7,wherein said culture medium lacks serum.
 10. The method of claim 7,wherein the undifferentiated neural cells are human cells.
 11. Themethod of claim 7, wherein the undifferentiated neural cells arecultured in a suspension culture.
 12. The method of claim 7, wherein theundifferentiated neural cells are cultured in an adherent culture.